Composition for inducing proliferation or accumulation of regulatory t cells

ABSTRACT

It was found that bacteria belonging to the genus  Clostridium  induce accumulation of regulatory T cells (Treg cells) in the colon. Moreover, the present inventors found that regulatory T cells (Treg cells) induced by from these bacteria suppressed proliferation of effector T-cells. From these findings, the present inventors found that the use of bacteria belonging to the genus  Clostridium  or a physiologically active substance derived therefrom made it possible to induce proliferation or accumulation of regulatory T cells (Treg cells), and further to suppress immune functions.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/492,850, filed Sep. 22, 2014, which is a continuation of U.S.application Ser. No. 13/701,467, filed Feb. 11, 2013, which is anational stage filing under 35 U.S.C. 371 of International ApplicationPCT/JP2011/063302, filed Jun. 3, 2011, which claims the benefit of andpriority to JP 2010-129134, filed Jun. 4, 2010 and PCT/JP2010/071746,filed Dec. 3, 2010. The entire teachings of the referenced applicationsare incorporated by reference herein.

TECHNICAL FIELD

The present invention relates to a composition which has an effect ofinducing proliferation or accumulation of regulatory T cells, and whichcomprises, as an active ingredient, bacteria belonging to the genusClostridium, a physiologically active substance derived from thebacteria, bacterial spores, or the like. The present invention alsorelates to a method for inducing proliferation or accumulation ofregulatory T cells, as well as a method for inhibiting suchproliferation or accumulation. Moreover, the present invention relatesto a vaccine composition containing at least one strain of bacteriabelonging to the genus Clostridium or a spore of bacteria, as well as amethod for treating or preventing at least one disease or conditionselected from infectious diseases and autoimmune diseases byadministering the vaccine composition to an individual in need thereof.The present invention also relates to a method for screening for acompound that promotes proliferation or accumulation of regulatory Tcells, as well as a non-human mammal which is used in this method, andin which a reporter gene is expressed under control of IL-10 geneexpression.

BACKGROUND ART

Hundreds of species of commensal microorganisms are harbored ingastrointestinal tracts of mammals, and intimately interact with thehost immune systems.

Results of researches using germ-free (GF) animals have shown that thecommensal microorganisms exert great influences on the development ofmucosal immune systems such as histogenesis of Peyer's patches (PPs) andisolated lymphoid follicles (ILFs), secretion of antimicrobial peptidesfrom epithelium, and accumulation of unique lymphocytes in mucosaltissues, the unique lymphocytes including immunoglobulin A-producingplasma cells, intraepithelial lymphocytes, IL-17-producing CD4-positiveT cells (Th 17), and IL-22-producing NK-like cells (Non-Patent Documents1 to 7). Consequently, the presence of intestinal bacteria enhancesprotective functions of the mucous membranes, providing the hosts withrobust immune responses against pathogenic microbes invading the bodies.On the other hand, the mucosal immune systems maintain unresponsivenessto dietary antigens and harmless microbes (Non-Patent Document 3). Forthis reason, abnormality in the regulation of cross-talk betweencommensal bacteria and an immune system (intestinal dysbiosis) may leadto overly robust immune response to environmental antigens, so thatinflammatory bowel disease (IBD) is caused (Non-Patent Documents 8 to10).

Results of Recent studies have shown that individual commensal bacteriacontrol differentiation of their specific immune cells in the mucosalimmune system. For example, Bacteroides fragilis, which is a commensalbacterium in humans, specifically induces a systemic Th1 cell responseand a mucosal IL-10-producing T cell response in mice, and plays a rolein protecting the host from colitis, which would otherwise be caused bya pathogen (Non-Patent Document 3). Segmented filamentous bacteria,which are intestinal commensal bacteria in mice, are shown to inducemucosal Th17 cell response and thereby to enhance resistance againstinfection of gastrointestinal tracts of the host with a pathogen(Non-Patent Documents 11 to 13). In addition, short-chain fatty acidsderived from several commensal bacteria are known to suppress intestinalinflammation (Non-Patent Document 14). Moreover, it is presumed that thepresence of some species of intestinal microbiota exerts a greatinfluence on the differentiation of regulatory T cells (hereafterreferred to as “Treg cells”) which maintain homeostasis of the immunesystem.

Meanwhile, regulatory T cells which have been identified as a subsetsuppressing immunity are CD4⁺ T cells in which a transcription factorFoxp3 is expressed, and are known to play an important role inmaintaining immunological homeostasis (Non-Patent Documents 8, 9, 15,and 16). Moreover, it has been known that the Foxp3-expressing cells arepresent in a large number especially in the colon, and only Treg cellspresent locally in the colon constantly expresses IL-10, which is animmunosuppressive cytokine, at a high level (Non-Patent Document 17). Itis also known that animals having CD4⁺ Foxp3⁺ cells from which IL-10 isspecifically removed develop inflammatory bowel disease (Non-PatentDocument 18).

Accordingly, if the mechanism of the induction of Treg cells whichproduce IL-10 in the colon at a high level is elucidated,immunosuppression can be enhanced, which in turn can be applied totreatment of autoimmune diseases such as inflammatory bowel disease, aswell as to organ transplantation.

However, mechanisms of how a large number of Treg cells come to bepresent in the colon and how the Treg cells produce IL-10 in the colonat a high level are still unclear. Moreover, it is also still unclearwhat species of bacteria constituting the intestinal commensal bacterialflora exerts the influence on the induction of regulatory T cells.

CITATION LIST Non Patent Literature

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SUMMARY OF INVENTION Technical Problem

The present invention has been made in view of the above-describedproblems of the conventional techniques. Accordingly, an object of thepresent invention is to identify intestinal commensal bacteria whichinduce the proliferation or accumulation of regulatory T cells. Anotherobject of the present invention is to provide compositions or the likewhich comprise the identified intestinal commensal bacteria or aphysiologically active substance derived therefrom, and which thus haveinduce the proliferation or accumulation of regulatory T cells (Tregcells).

Solution to Problem

The present inventors have made earnest studies to solve theabove-described problems. As a result, the present inventors have foundthat a chloroform-treated fraction and a spore-forming fraction of afecal sample obtained from a mammal induces accumulation of regulatory Tcells (Treg cells) in the colon. Moreover, the present inventors havefound that bacteria belonging to the genus Clostridium induceproliferation or accumulation of regulatory T cells in the colon. Thepresent inventors have also found that the regulatory T cells induced bythese bacteria suppress proliferation of effector T cells. Furthermore,the present inventors have also found that colonization of bacteriabelonging to the genus Clostridium and resultant proliferation oraccumulation of Treg cells regulate local and systemic immune responses.

From these findings, the present inventors have found that the use ofbacteria belonging to the genus Clostridium, spores thereof, or aphysiologically active substance derived therefrom makes it possible toinduce the proliferation or accumulation of regulatory T cells (Tregcells), and further to suppress immune functions.

More specifically, the present invention has the following aspects:

(1) A composition that induces proliferation or accumulation ofregulatory T cells, the composition comprising, as an active ingredient,at least one substance selected from the group consisting of thefollowing (a) to (c):

(a) bacteria belonging to the genus Clostridium or a physiologicallyactive substance derived from the bacteria;

(b) a spore-forming fraction of a fecal sample obtained from a mammal ora culture supernatant of the fraction; and

(c) a chloroform-treated fraction of a fecal sample obtained from amammal or a culture supernatant of the fraction.

(2) The composition according to claim 1, wherein

the regulatory T cells are transcription factor Foxp3-positiveregulatory T cells or IL-10-producing regulatory T cells.

(3) The composition according to any one of (1) and (2), wherein

the composition has an immunosuppressive effect.

(4) The composition according to any one of (1) to (3), wherein

the composition is a pharmaceutical composition.

(5) A method for inducing proliferation or accumulation of regulatory Tcells in an individual (e.g., an individual in need thereof, such as anindividual in need of induction of proliferation or accumulation ofregulatory T cells), the method comprising a step of administering, tothe individual, at least one substance selected from the groupconsisting of the following (a) to (c):

(a) bacteria belonging to the genus Clostridium or a physiologicallyactive substance derived from the bacteria;

(b) a spore-forming fraction of a fecal sample obtained from a mammal ora culture supernatant of the fraction; and

(c) a chloroform-treated fraction of a fecal sample obtained from amammal or a culture supernatant of the fraction.

(6) A method for inducing proliferation or accumulation of regulatory Tcells in an individual (e.g., an individual in need thereof, such as anindividual in need of induction of proliferation or accumulation ofregulatory T cells), the method comprising a step of administering anantibiotic against Gram-negative bacteria to the individual. And theantibiotic can be administered alone or in combination with at least onesubstance selected from the group consisting of the following (a) to(c):

(a) bacteria belonging to the genus Clostridium or a physiologicallyactive substance derived from the bacteria;

(b) a spore-forming fraction of a fecal sample obtained from a mammal ora culture supernatant of the fraction; and

(c) a chloroform-treated fraction of a fecal sample obtained from amammal or a culture supernatant of the fraction.

(7) A method for inducing proliferation or accumulation of regulatory Tcells in an individual, the method comprising a step of administering,to the individual, at least one substance selected from the groupconsisting of almond skin, inulin, oligofructose, raffinose, lactulose,pectin, hemicellulose, amylopectin, acetyl-Co A, biotin, beet molasses,yeast extracts, and resistant starch.(8) The method according to any one of (5) to (7), wherein

a therapeutic composition is further administered to the individual.

Note that, the “therapeutic composition” here is meant to be somethingother than (a)-(c) described in (5) and (6), the antibiotic againstGram-negative bacteria described in (6), or the substances described in(7).

(9) The method according to (8), wherein

the therapeutic composition is at least one composition selected fromthe group consisting of corticosteroids, mesalazine, mesalamine,sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs,cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate,antihistamines, glucocorticoids, epinephrine, theophylline, cromolynsodium, anti-leukotrienes, anti-cholinergic drugs for rhinitis,anti-cholinergic decongestants, mast-cell stabilizers, monoclonalanti-IgE antibodies, vaccines, and combinations thereof.

(10) The method according to any one of (5) to (9), wherein

one measurement selected from the group consisting of promotion of IL-10expression, promotion of CTLA4 expression, promotion of IDO expression,and suppression of IL-4 expression is used as an index of the inductionof proliferation or accumulation of regulatory T cells in theindividual.

(11) A method for inhibiting proliferation or accumulation of regulatoryT cells in an individual (e.g., an individual thereof), the methodcomprising a step of administering an antibiotic against Gram-positivebacteria to the individual.(12) The composition according to any one of (5) to (11), wherein

the regulatory T cells are transcription factor Foxp3-positiveregulatory T cells or IL-10-producing regulatory T cells.

(13) A vaccine composition comprising at least one substance selectedfrom the group consisting of the following (a) to (c):

(a) bacteria belonging to the genus Clostridium;

(b) a spore of bacteria in a spore-forming fraction of a fecal sampleobtained from a mammal; and

(c) bacteria in a chloroform-treated fraction of a fecal sample obtainedfrom a mammal.

(14) A method for treating aiding in treating, reducing the severity of,or preventing at least one disease selected from infectious diseases andautoimmune diseases in an individual (e.g., an individual in needthereof, such as an individual in need of treatment, reduction in theseverity of or prevention of at least one such disease), the methodcomprising administering the vaccine composition according to (13) tothe individual.(15) A method for screening for a compound having an activity to promoteproliferation or accumulation of regulatory T cells, the methodcomprising:

(I) preparing a test substance from at least one substance selected fromthe group consisting of the following (a) to (c):

-   -   (a) bacteria belonging to the genus Clostridium or a        physiologically active substance derived from the bacteria;    -   (b) a spore-forming fraction of a fecal sample obtained from a        mammal or a culture supernatant of the fraction; and    -   (c) a chloroform-treated fraction of a fecal sample obtained        from a mammal or a culture supernatant of the fraction.

(II) preparing non-human mammals in which a reporter gene is to beexpressed under control of IL-10 gene expression;

(III) bringing the test substance into contact with the non-humanmammal;

(IV) after the contact with the test substance, detecting cellsexpressing the reporter gene in a CD4⁺ Foxp3⁺ cell group of thenon-human mammal, and determining the number of cells in the CD4⁺ Foxp3⁺cell group expressing the reporter gene or a ratio of cells in the CD4⁺Foxp3⁺ cell group expressing the reporter gene to cells in the CD4⁺Foxp3⁺ cell group not expressing the reporter gene;

(V) detecting cells expressing the reporter gene in a CD4⁺ Foxp3⁺ cellgroup of the non-human mammal which has not been in contact with thetest substance, and determining the number of cells in the CD4⁺ Foxp3⁺cell group expressing the reporter gene or a ratio of cells in the CD4⁺Foxp3⁺ cell group expressing the reporter gene to cells in the CD4⁺Foxp3⁺ cell group not expressing the reporter gene; and

(VI) comparing the number or the ratio determined in (IV) with thenumber or the ratio determined in (V), and determining, when the numberor the ratio determined in (IV) is greater than that determined in (V),that the test substance is a compound that promotes proliferation oraccumulation of Treg cells.

(16) A non-human mammal which is used for the method according to (15),and in which the reporter gene is expressed under the control of theIL-10 gene expression.(17) A method for isolating, from a sample of bacteria belonging to thegenus Clostridium, a compound having an activity to promoteproliferation or accumulation of regulatory T cells, the methodcomprising (I) to (III):

(I) preparing a genomic DNA from the sample of bacteria belonging to thegenus Clostridium;

(II) inserting the genomic DNA into a cloning system, and preparing agene library derived from the sample of bacteria belonging to the genusClostridium; and

(III) isolating a compound having an activity to promote proliferationor accumulation of regulatory T cells, by use of the gene libraryobtained in step (II).

(18) A method of treatment comprising (I) to (III):

(I) measuring the percentage and/or absolute amounts of ClostridiumClusters IV and XIV in the microbiota of a subject,

(II) comparing them to the same measurements in a healthy individual;and

(III) administering a substance to the subject, if a statisticallysignificant decrease in the number/amounts of Clostridium cluster IV,XIV in the subject compared to the healthy individual is detected,wherein the substance is at least one substance selected from the groupconsisting of the following (a) to (c):

-   -   (a) the substance according to any one of claims 1 to 4    -   (b) an antibiotic against Gram-negative bacteria; and    -   (c) the substance according to claim 7.        (19) A method of monitoring, comprising (I) to (II):

(I) measuring level of Clostridium cluster IV, XIV in a subject afteradministering at least one substance selected from the group consistingof the following (a) to (c):

-   -   (a) the substance according to any one of claims 1 to 4    -   (b) an antibiotic against Gram-negative bacteria; and    -   (c) the substance according to claim 7; and

(II) if the level increases, it is judged to be a sign that the subjectis responding favorably.

Advantageous Effects of Invention

The compositions of the present invention containing as an activeingredient bacteria belonging to the genus Clostridium or aphysiologically active substance derived from the bacteria serves as anexcellent composition for inducing the proliferation or accumulation ofregulatory T cells (Treg cells). Immunity in a living organism can besuppressed through administration of the composition of the presentinvention as a pharmaceutical product or ingestion of the composition asa food or beverage. Accordingly, the composition of the presentinvention can be used, for example, to prevent or treat autoimmunediseases or allergic diseases, as well as to suppress immunologicalrejection in organ transplantation or the like. In addition, if a foodor beverage such as a health food comprises the composition of thepresent invention, healthy individuals can ingest the composition easilyand routinely. As a result, it is possible to induce the proliferationor accumulation of regulatory T cells and thereby to improve immunefunctions.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic diagram showing a method of producing Il10^(venus)mouse.

FIG. 2 is a diagram showing results of Southern blotting performed foranalysis as to whether or not the Il10^(venus) mice have an Il10^(venus)allele.

FIG. 3 is a FACS dot-plot diagram showing results obtained whenVenus-positive cells and Venus-negative cells from the Il10^(venus) micewere sorted.

FIG. 4 is a graph showing the results obtained when the amounts of IL-10mRNA expressed in Venus positive-cells and Venus-negative cells of theIl10^(venus) mice were analyzed by real-time RT-PCR.

FIG. 5 is a graph showing change in the ratio of Foxp3⁺ cells in CD4⁺lymphocytes of SPF mice.

FIG. 6 shows FACS dot-plot diagrams showing analysis results of theratios of Foxp3⁺ cells in CD4⁺ lymphocytes isolated from the smallintestine, the colon, and the peripheral lymph nodes of GF mice and SPFmice.

FIG. 7 is a graph showing analysis results of the ratios of Foxp3⁺ cellsin CD4⁺ lymphocytes isolated from the small intestine, the colon, andthe peripheral lymph nodes of GF mice and SPF mice.

FIG. 8 shows graphs showing analysis results of the numbers of CD4⁺Foxp3⁺ cells isolated from the small intestine, the colon, and theperipheral lymph nodes of GF mice and SPF mice.

FIG. 9 is a plot diagram showing analysis results of the ratios ofVenus⁺ cells in CD4⁺ cells in various tissues of SPF mice treated withantibiotics.

FIG. 10 shows FACS dot-plot diagrams showing analysis results of theratio of Foxp3⁺ cell in CD4⁺ lymphocytes isolated from the coloniclamina propria of GF mice to which a fecal suspension of SPF mice wasadministered.

FIG. 11 is a graph showing analysis results of the ratios of Foxp3⁺cells in CD4⁺ lymphocytes isolated from the lamina propria of the colonand the lamina propria of the small intestine of GF mice to which afecal suspension of SPF mice was administered.

FIG. 12 is a graph showing analysis results of the ratio of Foxp3⁺ cellsin CD4⁺ lymphocytes isolated from the lamina propria of mice deficientin ILFs, PPs, and colonic-patches.

FIG. 13 shows FACS dot-plot diagrams showing analysis results of theratios of Foxp3⁺ cells in CD4⁺ lymphocytes isolated from the coloniclamina propria of GF mice to which specific commensal bacteria wereadministered.

FIG. 14 shows graphs showing analysis results of the ratios of Foxp3⁺cells in CD4⁺ lymphocytes isolated from the colonic lamina propria of GFmice to which specific commensal bacteria were administered.

FIG. 15 is a graph showing analysis results of the ratios of IFN-γ⁺cells in CD4⁺ lymphocytes isolated from the colonic lamina propria ofmice in which specific commensal bacteria were colonized.

FIG. 16 is a graph showing analysis results of the ratios of IL-17⁺cells in CD4⁺ lymphocytes isolated from the colonic lamina propria ofmice in which specific commensal bacteria were colonized.

FIG. 17 is a graph showing analysis results of the ratios of Foxp3⁺cells in CD4⁺ lymphocytes isolated from the colon of kinds of SPF miceeach being deficient in a pathogen-associated molecular patternrecognition receptor-associated factor.

FIG. 18 is a graph showing analysis results of the ratios of Foxp3⁺cells in CD4⁺ lymphocytes isolated from the colonic lamina propria ofMyd88^(−/−) mice in which the Clostridium was colonized.

FIG. 19 shows FACS dot-plot diagrams showing analysis results of theratios of Venus⁺ cells in lymphocytes isolated from various tissues ofIl10^(venus) mice.

FIG. 20 is a FACS dot-plot diagram showing analysis results of theexpression of a T cell receptor β chain on cell surfaces of lymphocytesisolated from the colonic lamina propria of Il10^(venus) mice.

FIG. 21 shows FACS dot-plot diagrams showing analysis results of theexpression of IL-17, IL-4, and IFN-γ in lymphocytes isolated from thecolonic lamina propria of Il10^(venus) mice.

FIG. 22 shows graphs showing analysis results of the amounts of mRNAs ofIL-10, CTLA4, Foxp3, and GITR expressed in spleen Foxp3⁻CD4⁺ cells,spleen Foxp3⁺ CD4⁺ cells, colonic lamina propria Venus⁺ cells, and smallintestinal lamina propria Venus⁺ cells.

FIG. 23 shows FACS dot-plot diagrams showing analysis results of theexpression of CD4, Foxp3, and Venus in the lamina propria of the smallintestine and the lamina propria of the colon of GF Il10^(venus) miceand SPF Il10^(venus) mice.

FIG. 24 shows FACS dot-plot diagrams showing analysis results of theexpression of Venus and Foxp3 of CD4 cells in various tissues of SPFIl10^(venus) mice.

FIG. 25 shows FACS dot-plot diagrams showing analysis results of theexpression of Foxp3 and Venus in Il10^(venus) mice in which specificcommensal bacteria were colonized.

FIG. 26 is a graph showing analysis results of the expression of Foxp3and/or Venus of CD4⁺ cells in the small intestine of Il10_(venus) micein which specific commensal bacteria were colonized.

FIG. 27 is a graph showing analysis results of the expression of Foxp3and/or Venus of CD4⁺ cells in the colon of Il10^(venus) mice in whichspecific commensal bacteria were colonized.

FIG. 28 is a plot diagram showing analysis results of the ratios ofVenus⁺ cells in CD4⁺ cells isolated from various tissues of Il10^(venus)mice treated with antibiotics.

FIG. 29 is a graph showing analysis results of immunoregulatoryfunctions of CD4⁺ Venus⁺ cells from the colonic lamina propria of GFIl10^(venus) mice in which the genus Clostridium was colonized, CD4⁺Venus⁺ cells from the colonic lamina propria of SPF Il10^(venus) mice,and CD4⁺ GFP⁺ cells from the spleen of Foxp3^(eGFP) reporter mice.

FIG. 30 is a graph showing the results obtained when SPF B6 mice weretreated with polymyxin B or vancomycin for 4 weeks, and then analyzedfor the ratio of Foxp3⁺ cells in the CD4⁺ cell group.

FIG. 31 is a graph showing the results obtained when SPF mice-derivedchloroform-treated feces were orally administered to GF mice, and thenthe ratio of Foxp3⁺ cells in the CD4⁺ cell group was analyzed.

FIG. 32 is a graph showing the general results of flow cytometryanalysis on Helios expression in LP lymphocytes in the thymuses or thecolons of SPF mice, GF mice, Lactobacillus-colonized mice, orClostridium-colonized mice.

FIG. 33 shows plot diagrams showing representative results of flowcytometry analysis on CD4 expression, Foxp3 expression, and Heliosexpression in the LP lymphocytes in the thymuses or the colons of theSPF mice, the GF mice, the Lactobacillus-colonized mice, or theClostridium-colonized mice.

FIG. 34 is a graph showing the results obtained when the whole colonsderived from GF mice, Lactobacillus-colonized mice, orClostridium-colonized mice were cultured, and the culture supernatantsthereof were analyzed for the TGF-β1 concentration by ELISA.

FIG. 35 is a graph showing the results obtained when intestinalepithelial cells (IECs) derived from GF mice or Clostridium-colonizedmice were cultured, and the culture supernatants thereof were analyzedfor the TGF-β1 concentration by ELISA.

FIG. 36 is a graph showing the results obtained when splenic CD4⁺ Tcells were cultured together with an anti-CD3 antibody and with aculture supernatant of IECs isolated from GF mice or mice colonized with46 bacterial strains of the genus Clostridium (Clost.) in the presenceor absence of an anti-IGF-β, antibody, and the T cells were collected onday 5 of the culture and analyzed for Foxp3 expression by real-timeRT-PCR.

FIG. 37 is a graph showing the results obtained when C57BL/6 GF micewere orally inoculated with 46 bacterial strains of the genusClostridium (Clost.) or three bacterial strains of the genusLactobacillus (Lacto.), and IECs were collected three weeks after theinoculation and analyzed for the relative mRNA expression level of theMMP2 gene by real-time RT-PCR.

FIG. 38 is a graph showing the results obtained when C57BL/6 GF micewere orally inoculated with 46 bacterial strains of the genusClostridium (Clost.) or three bacterial strains of the genusLactobacillus (Lacto.), and IECs were collected three weeks after theinoculation and analyzed for the relative mRNA expression level of theMMP9 gene by real-time RT-PCR.

FIG. 39 is a graph showing the results obtained when C57BL/6 GF micewere orally inoculated with 46 bacterial strains of the genusClostridium (Clost.) or three bacterial strains of the genusLactobacillus (Lacto.), and IECs were collected three weeks after theinoculation and analyzed for the relative mRNA expression level of theMMP13 gene by real-time RT-PCR.

FIG. 40 is a graph showing the results obtained when C57BL/6 GF micewere orally inoculated with 46 bacterial strains of the genusClostridium (Clost.) or three bacterial strains of the genusLactobacillus (Lacto.), and IECs were collected three weeks after theinoculation and analyzed for the relative mRNA expression level of theIDO gene by real-time RT-PCR.

FIG. 41 is a graph showing the results obtained when control mice (SPF)and Clostridium-administered mice (SPF+Clost.) were treated with 2% DSS,observed and measured for the body weight loss, the hardness of stool,and bleeding for six days, and then evaluated numerically.

FIG. 42 is a photograph showing the state of the colons collected on day6 after the control mice (SPF) and the Clostridium-administered mice(SPF+Clost.) were treated with 2% DSS.

FIG. 43 shows photomicrographs showing the results obtained when thecontrol mice (SPF) and the Clostridium-administered mice (SPF+Clost.)were treated with 2% DSS, and the colons thereof were collected on day 6and analyzed histologically by HE staining.

FIG. 44 is a graph showing the results obtained when control mice (SPF)and Clostridium-administered mice (SPF+Clost.) were sensitized withoxazolone, and subsequently the inside of each rectum was treated with a1% oxazolone/50% ethanol solution, and the body weight loss wasmeasured.

FIG. 45 shows photomicrographs showing the results obtained when thecontrol mice (SPF) and the Clostridium-administered mice (SPF+Clost.)were sensitized with oxazolone, and subsequently the inside of eachrectum was treated with a 1% oxazolone/50% ethanol solution, and thecolons obtained by the treatment were analyzed histologically by HEstaining.

FIG. 46 is a graph showing the results obtained when control mice (SPF)and Clostridium-administered mice (SPF+Clost.) were immunized byadministering alum-absorbed ovalbumin (OVA) twice at a 2-week interval,and the sera were collected therefrom and analyzed for the concentrationof OVA-specific IgE in these sera by ELISA.

FIG. 47 is a graph showing the results obtained when the control mice(SPF) and the Clostridium-administered mice (SPF+Clost.) were immunizedby administering the alum-absorbed OVA twice at a 2-week interval, andsplenic cells were collected and analyzed for IL-4 production of thesesplenic cells by in-vitro OVA restimulation.

FIG. 48 is a graph showing the results obtained when the control mice(SPF) and the Clostridium-administered mice (SPF+Clost.) were immunizedby administering the alum-absorbed OVA twice at a 2-week interval, andthe splenic cells were collected and analyzed for IL-10 production ofthese splenic cells by the in-vitro OVA restimulation.

FIG. 49 is Phylogenetic tree constructed by the neighbor-joining methodwith the resulting sequences of the 41 strains of Clostridium and thoseof known bacteria obtained from Genbank database using Mega software.

FIG. 50 is histograms showing Foxp3 expression gated CD4 cells from GFmice (Germ-free mouse #1 and #2) or GF mice colonized with three strainsof Clostridium belonging to cluster IV (3 strains of Clost. mouse #1 and#2).

FIG. 51 is histograms showing Foxp3 expression by CD4 positivelymphocytes from GF mice (GF) or GF mice gavaged with chloroform-treatedhuman stool (GF+Chloro.).

FIG. 52 is a graph showing Foxp3 expression by CD4 positive lymphocytesfrom GF mice (GF) or GF mice gavaged with chloroform-treated human stool(GF+Chloro.).

FIG. 53 is a graph showing amounts of Clostridium and Bacteroides infeces of mice gavaged with chloroform-treated human stool

DESCRIPTION OF EMBODIMENTS Composition Having Effect of InducingProliferation or Accumulation of Regulatory T Cells

The present invention provides a composition that induces proliferationor accumulation of regulatory T cells, the composition comprising, as anactive ingredient, at least one substance selected from the groupconsisting of the following (a) to (c):

(a) bacteria belonging to the genus Clostridium or a physiologicallyactive substance derived from the bacteria;

(b) a spore-forming fraction of a fecal sample obtained from a mammal ora culture supernatant of the fraction; and

(c) a chloroform-treated fraction of a fecal sample obtained from amammal or a culture supernatant of the fraction.

In the present invention “regulatory T cells” mean T cells which have afunction of suppressing an abnormal or excessive immune response, andwhich play a role in immune tolerance. The regulatory T cells aretypically transcription factor Foxp3-positive CD4-positive T cells.However, the regulatory T cells of the present invention also includetranscription factor Foxp3-negative regulatory T cells, as long as theregulatory T cells are IL-10-producing CD4-positive T cells.

The meaning of the “induces proliferation or accumulation of regulatoryT cells” in the present invention includes an effect of inducing thedifferentiation of immature T cells into regulatory T cells, whichdifferentiation leads to the proliferation or the accumulation ofregulatory T cells. In addition, the meaning of the “inducesproliferation or accumulation of regulatory T cells” in the presentinvention includes in-vivo effects, in vitro effects, and ex vivoeffects. Accordingly, all of the following effects are included: aneffect of inducing in vivo proliferation or accumulation of regulatory Tcells through administration or ingestion of the bacteria belonging tothe genus Clostridium or the physiologically active substance or thelike derived from the bacteria; an effect of inducing proliferation oraccumulation of cultured regulatory T cells by causing the bacteriabelonging to the genus Clostridium or the physiologically activesubstance or the like derived from the bacteria to act on the culturedregulatory T cells; and an effect of inducing proliferation oraccumulation of regulatory T cells which are collected from a livingorganism and which are intended to be subsequently introduced into aliving organism, such as the organism from which they were obtained oranother organism, by causing the bacteria belonging to the genusClostridium or the physiologically active substance or the like derivedfrom the bacteria to act on the regulatory T cells. The effect ofinducing proliferation or accumulation of regulatory T cells can beevaluated, for example, as follows. Specifically, the bacteria belongingto the genus Clostridium or the physiologically active substance or thelike derived from the bacteria is orally administered to an experimentalanimal such as a germ-free mouse, then CD4-positive cells in the colonare isolated, and the ratio of regulatory T cells contained in theCD4-positive cells is measured by flow cytometry (refer to Example 7).

The regulatory T cells of which proliferation or accumulation is inducedby the composition of the present invention are preferably transcriptionfactor Foxp3-positive regulatory T cells or IL-10-producing regulatory Tcells.

The “bacteria belonging to the genus Clostridium,” which are the activeingredient in the composition of the present invention, is notparticularly limited as long as the bacteria have the effect of inducingproliferation or accumulation of regulatory T cells. The bacteriapreferably belong to the cluster XIVa or the cluster IV. One strain ofthe bacteria alone can be used for the composition of the presentinvention, but two or more strains of the bacteria can be used togetherfor the composition of the present invention. The use of multiplestrains of bacteria belonging to the cluster XIVa or the cluster IV incombination can bring about an excellent effect on regulatory T cells.In addition to the bacteria belonging to these clusters, bacteriabelonging to other clusters (for example, bacteria belonging to thecluster III) can also be used in combination. If more than one strain ofbacteria is used (e.g., one or more strain belonging to cluster XIVa,one or more strain belonging to cluster IV, one or more strain belongingto a cluster other than cluster XIVa or cluster IV, such as one or morestrain belonging to cluster III), the type and number of strains usedcan vary widely. The type and number to be used can be determined basedon a variety of factors (e.g., the desired effect, such as induction orinhibition of proliferation or accumulation of regulatory T cells; thedisease or condition to be treated, prevented or reduced in severity;the age or gender of the recipient) The strains can be present in asingle composition, in which case they will be consumed or ingestedtogether, or can be present in more than one composition (e.g., each canbe in a separate composition), in which case they can be consumedindividually or the compositions can be combined and the resultingcombination (combined compositions) consumed or ingested. Any number orcombination of strains that proves effective (e.g., any number from oneto 200, such as 1 to 100, 1 to 50, 1 to 40, 1 to 30, 1 to 20, 1 to 10, 1to 5 and any number therebetween) can be administered. In certainembodiments of the present invention, a combination of some or all ofthe 46 strains described in Document (Itoh, K., and Mitsuoka, T.Characterization of clostridia isolated from faeces of limited floramice and their effect on caecal size when associated with germ-freemice. Lab. Animals 19: 111-118 (1985)) is used. For example, at leastone, two or more, three, three or more, four, four or more, five, fiveor more, six, six or more or any other number of the 46 describedstrains, including 46 strains, can be used. They can be used incombination with one another and in combination with strains notdescribed in the cited reference (e.g, in combination with one or morestrains belonging to cluster III). Note that, the cluster of “bacteriabelonging to the genus Clostridium” can be identified, for example, asfollows. Specifically, the bacteria belonging to the genus Clostridiumare classified by PCR using a primer set consisting of SEQ ID NOs 64 and65 (for Clostridium spp. belonging to the cluster XIVa) or a primer setconsisting of SEQ ID NOs 66 and 67 (for Clostridium spp. belonging tothe cluster IV) (refer to Example 18). In addition, the bacteriabelonging to the genus Clostridium are classified by sequencing of 16SrRNA gene amplified using a primer set consisting of SEQ ID NOs 19 and20 (refer to Example 7).

Viable cells of the bacteria belonging to the genus Clostridium can beused for the composition of the present invention, and killed cellsthereof may also be used for the composition. In addition, from theviewpoint of stability to heat, resistance to antibiotics and the like,and long storage period, the bacteria belonging to the genus Clostridiumare preferably in the form of spore.

The meaning of the “physiologically active substance derived frombacteria belonging to the genus Clostridium” of the present inventionincludes substances contained in the bacteria, secretion products of thebacteria, and metabolites of the bacteria. Such a physiologically activesubstance can be identified by purifying an active component from thebacteria, a culture supernatant thereof, or intestinal tract contents inthe intestinal tract of a mouse in which only bacteria belonging to thegenus Clostridium are colonized by an already known purification method.

The active ingredient “spore-forming fraction of a fecal sample obtainedfrom a mammal” in the composition of the present invention is notparticularly limited, as long as the fraction includes spore-formingbacteria present in feces of a mammal, and has the effect of inducingproliferation or accumulation of regulatory T cells.

The active ingredient “chloroform-treated fraction of a fecal sampleobtained from a mammal” in the composition of the present invention isnot particularly limited, as long as the fraction is obtained bytreating feces of a mammal with chloroform (for example, 3% chloroform),and has the effect of inducing proliferation or accumulation ofregulatory T cells.

Note that the “mammal” in the present invention is not particularlylimited, and examples thereof include humans, mice, rats, cattle,horses, pigs, sheep, monkeys, dogs, and cats.

Meanwhile, when the “spore-forming fraction of a fecal sample obtainedfrom a mammal” or the “chloroform-treated fraction of a fecal sampleobtained from a mammal” is cultured in a medium, substances contained inthe bacteria, secretion products of the bacteria, metabolites of thebacteria are released from the bacteria and the like contained in thefraction. The meaning of the active ingredient “culture supernatant ofthe fraction” in the composition of the present invention includes suchsubstances, secretion products, and metabolites. The culture supernatantis not particularly limited, as long as the culture supernatant has theeffect of inducing proliferation or accumulation of regulatory T cells.Examples of the culture supernatant include a protein fraction of theculture supernatant, a polysaccharide fraction of the culturesupernatant, a lipid fraction of the culture supernatant, and alow-molecular weight metabolite fraction of the culture supernatant.

The composition of the present invention may be in the form of apharmaceutical composition, a food or beverage (which may also be ananimal feed), or a reagent used for an animal model experiment, thepharmaceutical composition, the food or beverage, and the reagent havingthe effect of inducing proliferation or accumulation of regulatory Tcells. An example of the present invention revealed that regulatory Tcells (Treg cells) induced by bacteria or the like belonging to thegenus Clostridium suppressed the proliferation of effector T-cells.Accordingly, the composition of the present invention can be usedsuitably as a composition having an immunosuppressive effect. Theimmunosuppressive effect can be evaluated, for example, as follows.Specifically, regulatory T cells isolated from an experimental animal,such as a mouse, to which the composition of the present invention isorally administered are caused to act on effector T-cells (CD4⁺ CD25⁻cells) isolated from the spleen, and then proliferation ability thereofis measured by using the intake amount of [³H]-thymidine as an index(refer to Example 14).

The composition of the present invention can be used, for example, as apharmaceutical composition for preventing or treating an autoimmunedisease such as chronic inflammatory bowel disease, systemic lupuserythematosus, rheumatoid arthritis, multiple sclerosis, or Hashimoto'sdisease, or an allergic disease such as pollenosis or asthma; apharmaceutical composition for suppressing rejection in organtransplantation or the like; a food or beverage for improving immunefunctions; or a reagent for suppressing the proliferation or function ofeffector T-cells.

More specific examples of target diseases of the composition of thepresent invention include autoimmune diseases, allergic diseases, andrejection in organ transplantations and the like, such as inflammatorybowel disease (IBD), ulcerative colitis, Crohn's disease, sprue,autoimmune arthritis, rheumatoid arthritis, Type I diabetes, multiplesclerosis, graft vs. host disease following bone marrow transplantation,osteoarthritis, juvenile chronic arthritis, Lyme arthritis, psoriaticarthritis, reactive arthritis, spondy loarthropathy, systemic lupuserythematosus, insulin dependent diabetes mellitus, thyroiditis, asthma,psoriasis, dermatitis scleroderma, atopic dermatitis, graft versus hostdisease, acute or chronic immune disease associated with organtransplantation, sarcoidosis, atherosclerosis, disseminatedintravascular coagulation, Kawasaki's disease, Grave's disease,nephrotic syndrome, chronic fatigue syndrome, Wegener's granulomatosis,Henoch-Schoenlejnpurpurea, microscopic vasculitis of the kidneys,chronic active hepatitis, uveitis, septic shock, toxic shock syndrome,sepsis syndrome, cachexia, acquired immunodeficiency syndrome, acutetransverse myelitis, Huntington's chorea, Parkinson's disease,Alzheimer's disease, stroke, primary biliary cirrhosis, hemolyticanemia, polyglandular deficiency type I syndrome and polyglandulardeficiency type II syndrome, Schmidt's syndrome, adult (acute)respiratory distress syndrome, alopecia, alopecia areata, seronegativearthopathy, arthropathy, Reiter's disease, psoriatic arthropathy,chlamydia, yersinia and salmonella associated arthropathy,spondyloarhopathy, atheromatous disease/arteriosclerosis, atopicallergy, food allergies, autoimmune bullous disease, pemphigus vulgaris,pemphigus foliaceus, pemphigoid, linear IgA disease, autoimmunehaemolytic anaemia, Coombs positive haemolytic anaemia, acquiredpernicious anaemia, juvenile pernicious anaemia, myalgicencephalitis/Royal Free Disease, chronic mucocutaneous candidiasis,giant cell arteritis, primary sclerosing hepatitis, cryptogenicautoimmune hepatitis, Acquired Immunodeficiency Disease Syndrome,Acquired Immunodeficiency Related Diseases, Hepatitis C, common variedimmunodeficiency (common variable hypogammaglobulinaemia), dilatedcardiomyopathy, fibrotic lung disease, cryptogenic fibrosing alveolitis,postinflammatory interstitial lung disease, interstitial pneumonitis,connective tissue disease associated interstitial lung disease, mixedconnective tissue disease associated lung disease, systemic sclerosisassociated interstitial lung disease, rheumatoid arthritis associatedinterstitial lung disease, systemic lupus erythematosus associated lungdisease, dermatomyositis/polymyositis associated lung disease, Sjogren'sdisease associated lung disease, ankylosing spondy litis associated lungdisease, vasculitic diffuse lung disease, haemosiderosis associated lungdisease, drug-induced interstitial lung disease, radiation fibrosis,bronchiolitis obliterans, chronic eosinophilic pneumonia, lymphocyticinfiltrative lung disease, postinfectious interstitial lung disease,gouty arthritis, autoimmune hepatitis, type-1 autoimmune hepatitis(classical autoimmune or lupoid hepatitis), type-2 autoimmune hepatitis(anti-LKM antibody hepatitis), autoimmune mediated hypoglycemia, type Binsulin resistance with acanthosis nigricans, hypoparathyroidism, acuteimmune disease associated with organ transplantation, chronic immunedisease associated with organ transplantation, osteoarthrosis, primarysclerosing cholangitis, idiopathic leucopenia, autoimmune neutropenia,renal disease NOS, glomerulonephritides, microscopic vasulitis of thekidneys, discoid lupus, erythematosus, male infertility idiopathic orNOS, sperm autoimmunity, multiple sclerosis (all subtypes),insulindependent diabetes mellitus, sympathetic ophthalmia, pulmonaryhypertension secondary to connective tissue disease, Goodpasture'ssyndrome, pulmonary manifestation of polyarteritis nodosa, acuterheumatio fever, rheumatoid spondylitis, Still's disease, systemicsclerosis, Takayasu's disease/arteritis, autoimmune thrombocytopenia,idiopathic thrombocytopenia, autoimmune thyroid disease,hyperthyroidism, goitrous autoimmune hypothyroidism (Hashimoto'sdisease), atrophic autoimmune hypothyroidism, primary myxoedema,phacogenic uveitis, primary vasculitis, vitiligo, allergic rhinitis(pollen allergies), anaphylaxis, pet allergies, latex allergies, drugallergies, allergic rhinoconjuctivitis, eosinophilic esophagitis,hypereosinophilic syndrome, eosinophilic gastroenteritis cutaneous lupuserythematosus, eosinophilic esophagitis, hypereosinophilic syndrome, andeosinophilic gastroenteritis.

The composition of the present invention can also be used as apharmaceutical composition for preventing or treating infectiousdiseases in an individual whose resistance to the infectious diseases isimpaired because of damage due to excessive inflammation caused by theimmunity.

Example of infectious pathogens which impair maintenance or recovery ofhomeostasis of a host, and which eventually bring about suchimmunopathological tissue damage include Salmonella, Shigella,Clostridium difficile, Mycobacterium (which cause the diseasetuberculosis), protozoa (which cause the disease malaria), filarialnematodes (which cause the disease filariasis), Schistosoma (which causethe disease schistosomiasis), Toxoplasma (which cause the diseasetoxoplasmosis), Leishmania (which cause the disease leishmaniasis), HCVand HBV (which cause the disease hepatitis C and hepatitis B), andherpes simplex viruses (which cause the disease herpes).

Pharmaceutical preparations can be formulated from the composition ofthe present invention by already known drug formulation methods. Forexample, the composition of the present invention can be used orally orparenterally in the forms of capsules, tablets, pills, liquids, powders,granules, fine granules, film-coated preparations, pellets, troches,sublingual preparations, chewables, buccal preparations, pastes, syrups,suspensions, elixirs, emulsions, liniments, ointments, plasters,cataplasms, transdermal absorption systems, lotions, inhalations,aerosols, injections, suppositories, and the like.

For formulating these preparations, the composition of the presentinvention can be used in appropriate combination with carriersacceptable pharmacologically or acceptable for a food or beverage,specifically, with sterile water, physiological saline, vegetable oil,solvent, a base material, an emulsifier, a suspending agent, asurfactant, a stabilizer, a flavoring agent, an aromatic, an excipient,a vehicle, a preservative, a binder, a diluent, a tonicity adjustingagent, a soothing agent, a bulking agent, a disintegrating agent, abuffer agent, a coating agent, a lubricant, a colorant, a sweetener, athickening agent, a flavor corrigent, a solubilizer, other additives, orthe like.

Meanwhile, for formulating a pharmaceutical preparation thereof, andparticularly for formulating a pharmaceutical preparation for oraladministration, it is preferable to use in combination a compositionwhich enables an efficient delivery of the composition of the presentinvention to the colon, from the viewpoint of more efficiently inducingthe proliferation or accumulation of regulatory T cells in the colon.

Such a composition or method which enables the delivery to the colon isnot particularly limited, and known compositions or methods can beemployed as appropriate. Examples thereof include pH sensitivecompositions, more specifically, enteric polymers which release theircontents when the pH becomes alkaline after the enteric polymers passthrough the stomach. When a pH sensitive composition is used forformulating the pharmaceutical preparation, the pH sensitive compositionis preferably a polymer whose pH threshold of the decomposition of thecomposition is 6.8 to 7.5. Such a numeric value range is a range wherethe pH shifts toward the alkaline side at a distal portion of thestomach, and hence is a suitable range for use in the delivery to thecolon.

Moreover, another example of the composition enabling the delivery tothe colon is a composition which ensures the delivery to the colon bydelaying the release of the contents by approximately 3 to 5 hours,which corresponds to the small intestinal transit time. In an example offormulating a pharmaceutical preparation using the composition fordelaying the release, a hydrogel is used as a shell. The hydrogel ishydrated and swells upon contact with gastrointestinal fluid, so thatthe contents are effectively released. Furthermore the delayed releasedosage units include drug-containing compositions having a materialwhich coats or selectively coats a drug. Examples of such a selectivecoating material include in vivo degradable polymers, graduallyhydrolyzable polymers, gradually water-soluble polymers, and/or enzymedegradable polymers. A preferred coating material for efficientlydelaying the release is not particularly limited, and examples thereofinclude cellulose-based polymers such as hydroxypropyl cellulose,acrylic acid polymers and copolymers such as methacrylic acid polymersand copolymers, and vinyl polymers and copolymers such aspolyvinylpyrrolidone.

Examples of the composition enabling the delivery to the colon furtherinclude bioadhesive compositions which specifically adhere to thecolonic mucosal membrane (for example, a polymer described in thespecification of U.S. Pat. No. 6,368,586), and compositions into which aprotease inhibitor is incorporated for protecting particularly abiopharmaceutical preparation in the gastrointestinal tracts fromdecomposition due to an activity of a protease.

An example of a system enabling the delivery to the colon is a system ofdelivering a composition to the colon by pressure change in such a waythat the contents are released by utilizing pressure change caused bygeneration of gas in bacterial fermentation at a distal portion of thestomach. Such a system is not particularly limited, and a more specificexample thereof is a capsule which has contents dispersed in asuppository base and which is coated with a hydrophobic polymer (forexample, ethyl cellulose).

Another example of the system enabling the delivery to the colon is asystem of delivering a composition to the colon, the system beingspecifically decomposed by an enzyme (for example, a carbohydratehydrolase or a carbohydrate reductase) present in the colon. Such asystem is not particularly limited, and more specific examples thereofinclude systems which use food components such as non-starchpolysaccharides, amylose, xanthan gum, and azopolymers.

When used as a pharmaceutical composition, the composition of thepresent invention may be used in combination with an already knownpharmaceutical composition for use in immunosuppression. Such a knownpharmaceutical composition is not particularly limited, and may be atleast one therapeutic composition selected from the group consisting ofcorticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazinederivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine,azathiopurine, prednisone, methotrexate, antihistamines,glucocorticoids, epinephrine, theophylline, cromolyn sodium,anti-leukotrienes, anti-cholinergic drugs for rhinitis, anti-cholinergicdecongestants, mast-cell stabilizers, monoclonal anti-IgE antibodies,vaccines (preferably vaccines used for vaccination where the amount ofan allergen is gradually increased), and combinations thereof. It ispreferable to use these therapeutic compositions in combination with thecomposition of the present invention.

When the composition of the present invention is used as a food orbeverage, the food or beverage can be, for example, a health food, afunctional food, a food for specified health use, a dietary supplement,a food for patients, or an animal feed. The food or beverage of thepresent invention can be ingested in the forms of the compositions asdescribed above, and also can be ingested in the forms of various foodsand beverages. Specific examples of the foods and beverages includevarious beverages such as juices, refreshing beverages, tea beverages,drink preparations, jelly beverages, and functional beverages; alcoholicbeverages such as beers; carbohydrate-containing foods such as rice foodproducts, noodles, breads, and pastas; paste products such as fish hams,sausages, paste products of seafood; retort pouch products such ascurries, food dressed with a thick starchy sauces, and Chinese soups;soups; dairy products such as milk, dairy beverages, ice creams,cheeses, and yogurts; fermented products such as fermented soybeanpastes, yogurts, fermented beverages, and pickles; bean products;various confectionery products such as Western confectionery productsincluding biscuits, cookies, and the like, Japanese confectioneryproducts including steamed bean-jam buns, soft adzuki-bean jellies, andthe like, candies, chewing gums, gummies, cold desserts includingjellies, creme caramels, and frozen desserts; instant foods such asinstant soups and instant soy-bean soups; microwavable foods; and thelike. Further, the examples also include health foods and beveragesprepared in the forms of powders, granules, tablets, capsules, liquids,pastes, and jellies. The composition of the present invention can beused for animals including humans. The animals, other than humans, arenot particularly limited, and the composition can be used for variouslivestock, poultry, pets, experimental animals, and the like. Specificexamples of the animals include pigs, cattle, horses, sheep, goats,chickens, wild ducks, ostriches, domestic ducks, dogs, cats, rabbits,hamsters, mice, rats, monkeys, and the like, but the animals are notlimited thereto.

Without wishing to be bound by theory, in the present invention,individuals in which the relative abundance of bacteria belonging to thegroup Firmicutes (the group to which the Clostridium clusters IV andXIVa belong) is large gain more body weight than individuals in whichthe relative abundance of bacteria belonging to the group Bacteroidetesis large. Accordingly, the composition of the present invention iscapable of conditioning absorption of nutrients and improving feedefficiency. From such a viewpoint, the composition of the presentinvention can be used for promoting body weight gain, or for an animalfeed good in feed efficiency.

Moreover, the addition of the composition of the present invention to anantibiotic-free animal feed makes it possible to increase the bodyweight of a subject that ingests the animal feed to a level equal to orhigher than those achieved by antibiotic-containing animal feeds, andalso makes it possible to reduce pathogenic bacteria in the stomach to alevel equal to those achieved by typical antibiotic-containing animalfeeds. Accordingly, the composition of the present invention can be usedfor an animal feed which does not need the addition of antibiotics.

In addition, unlike conventional bacteria (Lactobacillus andBifidobacteria) in commercial use which are not easy to incorporate intothe livestock production, the composition of the present invention inthe spore form can be pelletized, sprayed, or easily mixed with ananimal feed, and also can be added to drinking water.

The feeding of such an animal feed using the composition of the presentinvention is not particularly limited, and the animal feed may be fed toa subject at regular intervals in a selective manner, or may be fed fora certain period (for example, at its birth, during weaning, or when thesubject to be fed is relocated or shipped).

Moreover, from the above-described viewpoint, the composition of thepresent invention can be preferably used for malnourished humans. Inother words, also when the subject who ingests the composition is ahuman, the composition of the present invention can preferably be usedfor promoting the body weight gain, and enhancing the energy absorptionfrom foods.

The food or beverage of the present invention can be manufactured by amanufacturing technique which is well known in the technical field. Tothe food or beverage, one or more components (for example, a nutrient)which are effective for the improvement of an immune function by theimmunosuppressive effect may be added. In addition, the food or beveragemay be combined with another component or another functional foodexhibiting a function other than the function of the improvement of animmune function to thereby serve as a multi-functional food or beverage.

Moreover, the composition of the present invention can be preferablyincorporated into foods requiring a processing step which may destroyordinary probiotic strains. Specifically, most commercially usableprobiotic strains cannot be incorporated into foods which need to beprocessed by any one of a heat treatment, long term storage, a freezingtreatment, a mechanical stress treatment, and a high-pressure treatment(for example, extrusion forming or roll forming). On the other hand,because of an advantageous nature of forming spores, the composition ofthe present invention can be easily incorporated into such processedfoods.

For example, the composition of the present invention in the form ofspore can survive even in a dried food, and can remain living even afterbeing ingested. Likewise, the composition of the present invention canwithstand low-temperature sterilization processes, typically processesat a temperature in a range from 70° C. to the boiling point, bothinclusive. Thus, the composition of the present invention can beincorporated into all kinds of dairy products. Furthermore, thecomposition of the present invention can withstand long-term storage ofmany years; high-temperature processing such as baking and boiling;low-temperature processing such as freezing and cold storage; andhigh-pressure treatments such as extrusion forming and roll forming.

The foods which need to be processed under such harsh conditions are notparticularly limited, and examples thereof include foods which need tobe processed in a microwave oven to be edible (for example, oatmeal),foods which need to be baked to be edible (for example, muffin), foodswhich need to be subjected to a sterilization high-temperature treatmentfor a short period of time to be edible (for example, milk), and foodswhich need to be heated to be drinkable (for example, hot tea).

When the composition of the present invention is administered oringested, the amount thereof for the administration or ingestion isselected as appropriate depending on the age, body weight, symptoms,health conditions, of a subject, the kind of the composition (apharmaceutical product, a food or beverage, or the like), and the like.For example, the amount per administration or ingestion is generally0.01 mg/kg body weight to 100 mg/kg body weight, and preferably 1 mg/kgbody weight to 10 mg/kg body weight. The present invention also providesa method for suppressing the immunity of a subject, the method beingcharacterized in that the bacteria belonging to the genus Clostridium orthe physiologically active substance derived from the bacteria isadministered into or ingested by the subject as described above.

A product of the composition of the present invention (a pharmaceuticalproduct, a food or beverage, or a reagent) or a manual thereof may beprovided with a note stating that the product can be used to suppressthe immunity (including a note stating that the product has animmunosuppressive effect, and a note stating that the product has aneffect of suppressing the proliferation or function of effectorT-cells). Here, the “provision to the product or the manual thereof withthe note” means that the note is provided to a main body, a container, apackage, or the like of the product, or the note is provided to amanual, a package insert, a leaflet, or other printed matters, whichdisclose information on the product.

<Method for Inducing Proliferation or Accumulation of Regulatory TCells>

As described above, and as will be shown in Examples, the administrationof the composition of the present invention to an individual makes itpossible to induce proliferation or accumulation of regulatory T cellsin the individual. Thus, the present invention can provides a method forinducing proliferation or accumulation of regulatory T cells in anindividual, the method comprising a step of administering, to theindividual, at least one substance selected from the group consisting ofthe following (a) to (c):

(a) bacteria belonging to the genus Clostridium or a physiologicallyactive substance derived from the bacteria;

(b) a spore-forming fraction of a fecal sample obtained from a mammal ora culture supernatant of the fraction; and

(c) a chloroform-treated fraction of a fecal sample obtained from amammal or a culture supernatant of the fraction.

Note that, the “individual” in the present invention is not particularlylimited, and examples thereof include humans, various kinds oflivestock, poultry, pets, experimental animals, and the like. The“individual” may be in a healthy state or a diseased state.

Moreover, as will be shown in Example 5 to be described later,Gram-positive commensal bacteria play principal roles in theproliferation or accumulation of regulatory T cells. Accordingly, thepresent invention can also provide a method for inducing proliferationor accumulation of regulatory T cells in an individual, the methodcomprising a step of administering an antibiotic against Gram-negativebacteria to the individual.

In the present invention, the “antibiotic against Gram-negativebacteria” is not particularly limited, and examples thereof includeaminoglycoside antibiotics (amikacin, gentamicin, kanamycin, neomycin,netilmicin, tobramycin, and paromomycin), cephalosporin antibiotics(cefaclor, cefamandole, cefoxitin, cefprozil, cefuroxime, cefixime,cefdinir, cefditoren, cefoperazone, cefotaxime, ceftazidime, ceftibuten,ceftizoxime, ceftriaxone, and cefoxotin), sulfonamides, ampicillin, andstreptomycin. Without wishing to be bound by theory, the “antibioticagainst Gram-negative bacteria” according to the present invention ispreferably one which reduces Gram-negative bacteria, and contributes tothe colonization of Gram-positive bacteria.

Moreover, a prebiotic composition such as almond skin, inulin,oligofructose, raffinose, lactulose, pectin, hemicellulose (such asxyloglucan and alpha-glucans), amylopectin, and resistant starch whichare not decomposed in the upper gastrointestinal tract and promote thegrowth of intestinal microbes in the intestinal tract, as well as growthfactors such as acetyl-Co A, biotin, beet molasses, and yeast extracts,contribute to the proliferation of bacteria belonging to the genusClostridium. Accordingly, the present invention can also provide amethod for inducing proliferation or accumulation of regulatory T cellsin an individual, the method comprising a step of administering, to theindividual, at least one substance selected from the group consisting ofthese substances.

Meanwhile, in the “method for inducing proliferation or accumulation ofregulatory T cells” of the present invention, the composition of thepresent invention, the above-described “antibiotic against Gram-negativebacteria,” and the above-described “prebiotic composition or growthfactor” may be used in combination. Such combined use is notparticularly limited, and examples of the combined use are as follows:the “antibiotic against Gram-negative bacteria” is administered to anindividual in advance, and then the composition of the present inventionis administered; the “antibiotic against Gram-negative bacteria” and thecomposition of the present invention are simultaneously administered toan individual; the “prebiotic composition or growth factor” isadministered to an individual in advance, and then the composition ofthe present invention is administered; the “prebiotic composition orgrowth factor” and the composition of the present invention aresimultaneously administered to an individual; the composition of thepresent invention, the “antibiotic against Gram-negative bacteria,” andthe “prebiotic composition or growth factor” are administered to anindividual simultaneously or individually at any appropriate time.

Moreover, a therapeutic composition may be administered to an individualtogether with at least one substance selected from the group consistingof the composition of the present invention, the “antibiotic againstGram-negative bacteria,” and the “prebiotic composition or growthfactor.”

Such a therapeutic composition is not particularly limited, and may beat least one therapeutic composition selected from the group consistingof corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazinederivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine,azathiopurine, prednisone, methotrexate, antihistamines,glucocorticoids, epinephrine, theophylline, cromolyn sodium,anti-leukotrienes, anti-cholinergic drugs for rhinitis, anti-cholinergicdecongestants, mast-cell stabilizers, monoclonal anti-IgE antibodies,vaccines (preferably, vaccines used for vaccination where the amount ofan allergen is gradually increased), and combinations thereof. It ispreferable to use these therapeutic compositions in combination with theabove-described substance.

Moreover, there is no particular limitation imposed on the combined useof the therapeutic composition with at least one substance selected fromthe group consisting of the composition of the present invention, the“antibiotic against Gram-negative bacteria,” and the “prebioticcomposition or growth factor”. For example, the “one substance” and thetherapeutic composition are administered orally or parenterally to anindividual simultaneously or individually at any appropriate time.

Moreover, in the above-described “method for inducing proliferation oraccumulation of regulatory T cells,” whether or not the administrationof the composition of the present invention or the like actually inducesthe proliferation or accumulation of regulatory T cells can bedetermined by using, as an index, increase or reinforcement of at leastone selected from the group consisting of the number of regulatory Tcells, the ratio of regulatory T cells in the T cell group of the colon,a function of regulatory T cells, and expression of a marker ofregulatory T cells. It is preferable to use one measurement selectedfrom the group consisting of promotion of IL-10 expression, promotion ofCTLA4 expression, promotion of IDO expression, and suppression of IL-4expression, as the index of the induction of proliferation oraccumulation of regulatory T cells.

Note that examples of a method for detecting such expression include thenorthern blotting, the RT-PCR, and the dot blotting for detection ofgene expression at the transcription level; and the ELISA, theradioimmunoassay, the immunoblotting, the immunoprecipitation, and theflow cytometry for detection of gene expression at the translationlevel.

Meanwhile, a sample used for measuring such an index is not particularlylimited, and examples thereof include blood sampled from an individualand tissue pieces obtained in a biopsy.

<Method for Predicting Response of Individual to Composition of PresentInvention and/or Prognosis of Individual>

The present invention can provide a method in which the absolute amountor the ratio of bacteria belonging to the genus Clostridium in amicrobiota of an individual is determined, and, when the ratio or theabsolute value of the bacteria belonging to the genus Clostridium isreduced in comparison with a base line value obtained by performing asimilar determination on an individual in a typical health state, it isdetermined that the individual is possibly responsive to the compositionof the present invention.

In one embodiment, a method to predict a subject's response to asubstance and/or the subject's prognosis is provided. The methodcomprises measuring the percentage or absolute amounts of Clostridiumclusters IV and XIV in the microbiota of the subject and comparing themto a baseline value of the same measurements in a prototypical healthysubject, wherein a decreased absolute amount or percentage level ofClostridium clusters IV and/or XIV indicates that the subject mayrespond favorably to the compositions of the invention.

In one embodiment, the method further comprises measuring thecomposition of the microbiota of the subject after administration of thesubstance, wherein an increase in the percentage or absolute number ofClostridium spp. belonging to clusters IV, XIV after administration ofthe compositions of the present invention relative to prior to theadministering is a positive indicator of enhanced immunosuppression (orimmunoregulation). The measurement of the composition of the subject'smicrobiota can be made with techniques known in the art, such as 16srRNAsequencing.

Note that, in these embodiments, the substance is at least one substanceselected from the group consisting of the following (a) to (e):

(a) bacteria belonging to the genus Clostridium or a physiologicallyactive substance derived from the bacteria;

(b) a spore-forming fraction of a fecal sample obtained from a mammal ora culture supernatant of the fraction;

(c) a chloroform-treated fraction of a fecal sample obtained from amammal or a culture supernatant of the fraction;

(d) an antibiotic against Gram-negative bacteria according to thepresent invention; and

(e) at least one substance selected from the group consisting of almondskin, inulin, oligofructose, raffinose, lactulose, pectin, hemicellulose(such as xyloglucan and alpha-glucans), amylopectin, acetyl-Co A,biotin, beet molasses, yeast extracts, and resistant starch.

<Method for Inhibiting Proliferation or Accumulation of Regulatory TCells>

As will be shown in Example 5 to be described later, Gram-positivecommensal bacteria have principal roles in the proliferation oraccumulation of regulatory T cells. Accordingly, the present inventioncan also provide a method for inhibiting proliferation or accumulationof regulatory T cells in an individual, the method comprising a step ofadministering an antibiotic against Gram-positive bacteria to theindividual.

In the present invention, the “antibiotic against Gram-positivebacteria” is not particularly limited, and examples thereof includecephalosporin antibiotics (cephalexin, cefuroxime, cefadroxil,cefazolin, cephalothin, cefaclor, cefamandole, cefoxitin, cefprozil, andceftobiprole); fluoroquinolone antibiotics (cipro, Levaquin, floxin,tequin, avelox, and norflox); tetracycline antibiotics (tetracycline,minocycline, oxytetracycline, and doxycycline); penicillin antibiotics(amoxicillin, ampicillin, penicillin V, dicloxacillin, carbenicillin,vancomycin, and methicillin); and carbapenem antibiotics (ertapenem,doripenem, imipenem/cilastatin, and meropenem).

As described above, the “individual” in the present invention is notparticularly limited, and examples thereof include humans, various kindsof livestock, poultry, pets, experimental animals, and the like. The“individual” may be in a healthy state or a diseased state. Such adiseased state is not particularly limited, and examples thereof includestates of being subjected to cancer immunotherapy and of suffering froman infectious disease.

Moreover, as another mode of the “method for inhibiting proliferation oraccumulation of regulatory T cells,” the present invention can provide amethod for inhibiting proliferation or accumulation of regulatory Tcells in an individual, the method comprising a step of administering,to the individual, any one of an antibody, an antibody fragment, and apeptide, which are against an antigen that is at least one substanceselected from the group consisting of the following (a) to (c):

(a) bacteria belonging to the genus Clostridium or a physiologicallyactive substance derived from the bacteria;

(b) a spore-forming fraction of a fecal sample obtained from a mammal ora culture supernatant of the fraction; and

(c) a chloroform-treated fraction of a fecal sample obtained from amammal or a culture supernatant of the fraction.

<Vaccine Composition and Method for Treating or Preventing InfectiousDisease or Autoimmune Disease by Using the Vaccine Composition>

As described above, and as will be shown in Example 15 to be describedlater, the induction of Treg cells in the colon by the Clostridium hasan important role in local and systemic immune responses. Accordingly,the present invention can also provide a “vaccine composition comprisingat least one substance selected from the group consisting of thefollowing (a) to (c): (a) bacteria belonging to the genus Clostridium;(b) a spore of bacteria in a spore-forming fraction of a fecal sampleobtained from a mammal; and (c) bacteria in a chloroform-treatedfraction of a fecal sample obtained from a mammal” and a “method fortreating, aiding in treating, reducing the severity of, or preventing atleast one disease selected from infectious diseases and autoimmunediseases in an individual, the method comprising administering thevaccine composition to the individual.”

Note that such “autoimmune diseases” are not particularly limited, andexamples thereof include those described as the “specific examples oftarget diseases” in <Composition Having Effect of Inducing Proliferationor Accumulation of Regulatory T cells>. The “infectious diseases” arealso not particularly limited, and examples thereof include infectiousdiseases associated with “infectious pathogens” described as the“example of infectious pathogens” in <Composition Having Effect ofInducing Proliferation or Accumulation of Regulatory T cells>.

<Method for Screening for Compound Having Activity to PromoteProliferation or Accumulation of Regulatory T Cells>

The present invention can also provide a method for screening for acompound having an activity to promote proliferation or accumulation ofregulatory T cells, the method comprising:

(1) preparing a test substance from at least one substance selected fromthe group consisting of the following (a) to (c):

-   -   (a) bacteria belonging to the genus Clostridium or a        physiologically active substance derived from the bacteria;    -   (b) a spore-forming fraction of a fecal sample obtained from a        mammal or a culture supernatant of the fraction; and    -   (c) a chloroform-treated fraction of a fecal sample obtained        from a mammal or a culture supernatant of the fraction.

(2) preparing non-human mammals in which a reporter gene is to beexpressed under control of IL-10 gene expression;

(3) bringing the test substance into contact with the non-human mammal;

(4) after the contact with the test substance, detecting cellsexpressing the reporter gene in a CD4⁺ Foxp3⁺ cell group of thenon-human mammal, and determining the number of cells in the CD4⁺ Foxp3⁺cell group expressing the reporter gene or a ratio of cells in the CD4⁺Foxp3⁺ cell group expressing the reporter gene to cells in the CD4⁺Foxp3⁺ cell group not expressing the reporter gene;

(5) detecting cells expressing the reporter gene in a CD4⁺ Foxp3⁺ cellgroup of the non-human mammal which has not been in contact with thetest substance, and determining the number of cells in the CD4⁺ Foxp3⁺cell group expressing the reporter gene or a ratio of cells in the CD4⁺Foxp3⁺ cell group expressing the reporter gene to cells in the CD4⁺Foxp3⁺ cell group not expressing the reporter gene; and

(6) comparing the absolute numbers or the ratios determined in steps (4)with the number or the ratio determined in (5), and determining, whenthe number or the ratio determined in (4) is greater than thatdetermined in (5), that the test substance is a compound that promotesproliferation or accumulation of Treg cells.

The “test substance” according to the present invention is notparticularly limited, as long as the test substance is a substanceprepared from at least one substance selected from the group consistingof the substances (a) to (c). Examples of the test substance includeproteins, polysaccharides, lipids, and nucleic acids which are derivedfrom at least one substance selected from the group consisting of theabove described substances (a) to (c).

The “non-human mammal in which a reporter gene is to be expressed undercontrol of IL-10 gene expression” according to the present invention isnot particularly limited, as long as the non-human mammal is a non-humanmammal having a reporter gene whose expression is controlled by an IL-10gene expression control region (for example, a promoter, or anenhancer). Examples of such a reporter gene include genes encodingfluorescent proteins (for example, GFP), and genes encoding luciferase.As the “non-human mammal in which a reporter gene is to be expressedunder control of IL-10 gene expression” according to the presentinvention, an Il10^(venus) mouse to be shown later in Examples can bepreferably used.

The “contact” according to the present invention is not particularlylimited, and examples thereof include administration of the testsubstance to the non-human mammal orally or parenterally (for example,intraperitoneal injection, or intravenous injection).

The present invention can also provide a non-human mammal which is usedfor the method, and in which the reporter gene is to be expressed underthe control of the IL-10 gene expression.

Furthermore, the present invention can also provide a method forisolating, from a sample of bacteria belonging to the genus Clostridium,a compound having an activity to promote proliferation or accumulationof regulatory T cells, the method comprising the following steps (1) to(3):

(1) preparing a genomic DNA from the sample of bacteria belonging to thegenus Clostridium;

(2) inserting the genomic DNA into a cloning system, and preparing agene library derived from the sample of bacteria belonging to the genusClostridium; and

(3) isolating a compound having an activity to promote proliferation oraccumulation of regulatory T cells, by use of the gene library obtainedin step (2).

In such steps, methods for the preparation and the isolation are notparticularly limited, and known techniques for an in-vitro or in-vivosystem can be used as appropriate. Moreover, the compound isolated bythis method is not particularly limited, and examples thereof includenucleic acids (for example, a DNA, a mRNA, and a rRNA) derived frombacteria belonging to the genus Clostridium, as well as polypeptides andproteins derived from the bacteria belonging to the genus Clostridium.

Other Embodiment Modes According to Present Invention

In addition to the above-described embodiment modes, the presentinvention can also provide the following embodiment modes.

Specifically, the present invention can also provide a method fordetermining the composition of a microbiota in an individual, whereinthe increase in the ratio or the absolute number of bacteria belongingto the genus Clostridium after the administration of the composition ofthe present invention to the individual with respect to the ratio or theabsolute number before the administration is used as an index ofincreased immunosuppression. In such a method, the method fordetermining the composition of the microbiota is not particularlylimited, and known techniques (for example, 16S rRNA sequencing) can beused as appropriate.

The present invention can also provide a method for measuringdifferentiation of Treg cells, wherein the increase in differentiationof Treg cells in an individual after administration of the compositionof the present invention to the individual with respect to that beforethe administration is used as an index of increased immunosuppression(or immunoregulation).

Moreover, the composition of the present invention can also beadministered to an individual under an antibiotic treatment. The timingof the administration is not particularly limited, and the compositionof the present invention can be administered before or simultaneouslywith the antibiotic treatment, for example. Meanwhile, the compositionof the present invention is preferably administered in the spore formfrom the viewpoint of resistance to antibiotics.

Moreover, in a preferred mode of such administration, the composition ofthe present invention is administered after or simultaneously withadministration of an antibiotic against Gram-positive bacteria, forexample. Note that such an “antibiotic against Gram-positive bacteria”is not particularly limited, and examples thereof include cephalosporinantibiotics (cephalexin, cefuroxime, cefadroxil, cefazolin, cephalothin,cefaclor, cefamandole, cefoxitin, cefprozil, and ceftobiprole);fluoroquinolone antibiotics (cipro, Levaquin, floxin, tequin, avelox,and norflox); tetracycline antibiotics (tetracycline, minocycline,oxytetracycline, and doxycycline); penicillin antibiotics (amoxicillin,ampicillin, penicillin V, dicloxacillin, carbenicillin, vancomycin, andmethicillin); and carbapenem antibiotics (ertapenem, doripenem,imipenem/cilastatin, and meropenem).

Meanwhile, in another preferred mode of such administration, thecomposition of the present invention is administered after (orsimultaneously with) a treatment using vancomycin, metronidazole,linezolid, ramoplanin, or fidaxomicin, for example.

EXAMPLES

Hereinafter, the present invention is described more specifically on thebasis of Examples. However, the present invention is not limited toExamples below.

Note that mice used in Examples were prepared or produced as follows. Inthe following description, mice may be referred to with “SPF” or “GF”attached in front thereof. These “SPF” and “GF” indicate that the micewere maintained in the absence of specific pathogenic bacteria (specificpathogen-free, SPF), and that the mice were maintained under Germ-Free(GF) conditions, respectively.

<Mice>

C57BL/6, Balb/c, and IQI mice maintained under SPF or GF conditions werepurchased from Sankyo Labo Service Corporation, Inc. (Japan), JAPAN SLC,INC. (Japan), CLEA Japan, Inc. (Japan), or The Jackson Laboratory (USA).GF mice and gnotobiotic mice were bread and maintained within thegnotobiotic facility of The University of Tokyo, Yakult CentralInstitute for Microbiological Research, or Sankyo Labo ServiceCorporation, Inc. Myd88^(−/−), Rip2^(−/−), and Card9^(−/−) mice wereproduced as described in Non-Patent Documents 1 to 3, and backcrossedfor 8 generations or more, so that a C57BL/6 genetic background wasachieved. Foxp3^(eGFP) mice were purchased from the Jackson Laboratory.

<Il10^(venus) Mice>

To form a bicistronic locus encoding both Il10 and Venus under controlof an Il10 promoter, a targeting construct was first created.Specifically, a cassette (IRES-Venus-SV40 polyA signal cassette, referto Non-Patent Document 4) which was made of an internal ribosome entrysite (IRES), a yellow fluorescent protein (Venus), and a SV40 polyAsignal (SV40 polyA) and which was arranged next to a neomycin-resistantgene (neo), was inserted between a stop codon and a polyA signal (Exon5) of a Il10 gene. Next, the obtained targeting construct was used tocause homologous recombination with the Il10 gene region in the genomeof mice. Thus, Il10_(venus) mice having an Il10^(venus) alleles wereproduced (refer to FIG. 1). Note that in FIG. 1 “tk” represents a genecoding thymidine kinase, “neo” represents the neomycin-resistant gene,and “BamH1” represents a cleavage site by the restriction enzyme BamH1.

Genomic DNAs were extracted from the Il10^(venus) mice, treated withBamH1, and Southern blotted by use of a probe shown in FIG. 1. FIG. 2shows the obtained results. Wild-type and Il10_(venus) alleles weredetected as bands having sizes of 19 kb and 5.5 kb, respectively. Hence,as is apparent from the results shown in FIG. 2, it was found that thehomologous recombination shown in FIG. 1 occurred in the genome of theIl10^(venus) mice.

Further, CD4⁺ Venus⁻ cells or CD4⁺ Venus⁺ cells in the colonic laminapropria of the Il10^(venus) mice were sorted by use of a FACSAria. Then,real-time RT-PCR was carried out on an ABI 7300 system by a method to bedescribed later, to determine the amount of IL-10 mRNA expressed. FIGS.3 and 4 show the obtained results. As is apparent from the results shownin FIGS. 3 and 4, it was found that, since the development of the IL-10mRNA was detected only in the CD4⁺ Venus⁺ cells, the expression of IL-10mRNA in the Il10^(venus) mice was correctly reflected in the expressionof Venus. Note that the germ-free states of such Il10^(venus) mice wereestablished in Central Institute for Experimental Animals (Kawasaki,Japan). The Il10^(venus) mice in the germ-free states were maintained invinyl isolators in Sankyo Labo Service Corporation, Inc. (Tokyo, Japan),and used in the following Examples.

Meanwhile, experiments and analyses in Examples were carried out asfollows.

<Method for Colonization of Mice with Bacteria and Analysis Thereof>

According to the description in Non-Patent Documents 5 and 6, mice inwhich SFB or Clostridium were colonized were produced. Cecal contents orfeces of the obtained gnotobiotic mice were dissolved in sterile wateror an anaerobic dilution solution. The dissolved cecal contents or fecesas they were or after a chloroform treatment were orally administered toGF mice. Three strains of the Lactobacillus and 16 strains of theBacteroides were cultured separately from each other in a BL or EG agarmedium in an anaerobic manner. The cultured bacteria were harvested,suspended in an anaerobic TS broth, and orally administrated forcibly toGF mice. The state of the colonization of the bacteria in the mice wasassessed by microscopic observation conducted on a smear preparation offecal pellets.

<Cell Separation and Flow Cytometry>

In order to isolate lymphocytes from the colonic lamina propria and thesmall intestinal lamina propria, the small intestine and the colon werecollected, and cut open longitudinally. Then, fecal content and the likethereinside were washed to remove. Subsequently, the small intestine andthe colon were shaken in HBSS containing 5 mM of EDTA at 37° C. for 20minutes. After removal of epithelium and fat tissue, the intestinaltissues were cut into small pieces. To the small pieces, RPMI 1640 (4%fetal bovine serum (FBS), 1 mg/ml of collagenase D, 0.5 mg/ml ofdispase, and 40 μg/ml of DNaseI (all of which were manufactured by RocheDiagnostics K.K.)) were added, and the mixture was shaken in a waterbath kept at 37° C. for 1 hour. The digested tissues were washed withHBSS containing 5 mM of EDTA, and resuspended in 5 ml of 40% percoll (GEHealthcare). The suspension was overlayered on 2.5 ml of 80% percoll ina 15-ml Falcon tube. Then, centrifugation was carried out at roomtemperature and at 2000 rpm for 20 minutes to conduct cell separation bypercoll density gradient centrifugation. Cells at the interface werecollected, and used as lamina propria lymphocytes. The collected cellswere suspend in a staining buffer (PBS, 2% FBS, 2 mM EDTA, and 0.09%NaN₃), and stained by use of an anti-CD4 antibody (RM4-5, BDBiosciences) labeled with PE or PE-Cy7. After the staining of CD4, Foxp3in the cells were stained by use of Cytofix/Cytoperm Kit Plus withGolgistop (BD Biosciences) or Foxp3 Staining Buffer Set (eBioscience),as well as an anti-Foxp3 antibody (FJK-16s, eBioscience) labeled withAlexa647. Flow cytometry was performed by use of a FACScant II, and thedata were analyzed by FlowJo software (TreeStar Inc.). The sorting ofthe cells were performed by use of a FACSAria.

<Real-Time RT-PCR>

From an RNA prepared by using RNeasy Mini Kit (Qiagen), a cDNA wassynthesized by use of a MMV reverse transcriptase (Promega KK). Theobtained cDNA was analyzed by real-time RT-PCR using Power SYBR GreenPCR Master Mix (Applied Biosystems) and ABI 7300 real time PCR system(Applied Biosystems), or real-time RT-PCR using SYBR Premix Ex Taq(TAKARA) and Light Cycler 480. For each sample, a value obtained wasnormalized for the amount of GAPDH. A primer set was designed by usingPrimer Express Version 3.0 (Applied Biosystems), and those exhibiting a90% or higher sequence identity at an initial evaluation were selected.The primer set used was as follows:

Foxp3 (SEQ ID NO: 1) 5′-GGCAATAGTTCCTTCCCAGAGTT-3′ (SEQ ID NO: 2)5′-GGGTCGCATATTGTGGTACTTG-3′ CTLA4 (SEQ ID NO: 3)5′-CCTTTTGTAGCCCTGCTCACTCT-3′ (SEQ ID NO: 4) 5′-GGGTCACCTGTATGGCTTCAG-3′GITR (SEQ ID NO: 5) 5′-TCAGTGCAAGATCTGCAAGCA-3′ (SEQ ID NO: 6)5′-ACACCGGAAGCCAAACACA-3′ IL-10 (SEQ ID NO: 7)5′-GATTTTAATAAGCTCCAAGACCAAGGT-3′ (SEQ ID NO: 8)5′-CTTCTATGCAGTTGATGAAGATGTCAA-3′ GAPDH (SEQ ID NO: 9)5′-CCTCGTCCCGTAGACAAAATG-3′ (SEQ ID NO: 10) 5′-TCTCCACTTTGCCACTGCAA-3′Mmp2 (SEQ ID NO: 11) 5′-GGACATTGTCTTTGATGGCA-3′ (SEQ ID NO: 12)5′-CTTGTCACGTGGTGTCACTG-3′ Mmp9 (SEQ ID NO: 13)5′-TCTCTGGACGTCAAATGTGG-3′ (SEQ ID NO: 14) 5′-GCTGAACAGCAGAGCCTTC-3′'Mmp13 (SEQ ID NO: 15) 5′-AGGTCTGGATCACTCCAAGG-3′ (SEQ ID NO: 16)5′-TCGCCTGGACCATAAAGAA-3′ Ido1 (SEQ ID NO: 17)5′-AGAGGATGCGTGACTTTGTG-3′ (SEQ ID NO: 18) 5′-ATACAGCAGACCTTCTGGCA-3′.

<Preparation and Culturing of Large Intestinal Epithelial Cells (IECs)>

First, the colon was collected, cut open longitudinally, and rinsed withPBS. Subsequently, the colon was treated with 1 mM dithiothreitol (DTT)at 37° C. for 30 minutes on a shaker, and then vortexed for one minuteto disrupt the epithelial integrity. The released IECs were collected,and suspended in 5 ml of 20% percoll. The suspension was overlayered on2.5 ml of 80% percoll in a 15-ml Falcon tube. Then, the tube wascentrifuged at 25° C. and 780 g for 20 minutes to conduct cellseparation by percoll density gradient centrifugation. Cells at theinterface were collected, and used as colonic IECs (purity: 90% orhigher, viability: 95%). The obtained IECs thus collected were suspendedin RPMI containing 10% FBS, and 1×10⁵ cells of the IECs were cultured ina 24-well plate for 24 hours. Thereafter, the culture supernatant wascollected, and measured for active TGF-β1 level by ELISA (Promega).

Meanwhile, for culturing T cells in vitro, 1.5×10⁵ MACS-purified splenicCD4⁺ T cells were cultured in each well of a round-bottomed 96-wellplate, together with a 50% conditioned medium in which IECs isolatedfrom GF mice or Clostridium-colonized mice were cultured, and with 25ng/ml of hIL-2 (Peprotech), in the presence or absence of 25 μg/ml of ananti-TGF-β antibody (R&D). Note that 10 μg/ml of an anti-CD3 antibodyand an anti-CD28 antibody (BD Bioscience) were bound to theround-bottomed plate. After a 5-day culture, the CD4⁺ T cells werecollected, and subjected to a real-time PCR.

<Colitis Experimental Model>

A fecal suspension of Clostridium-colonized mice was orally administeredto C57BL/6 mice (2-week old), and grown in a conventional environmentfor six weeks.

For preparing a DSS-induced colitis model, 2% (wt/vol) DSS (reagentgrade, DSS salt, molecular weight=36 to 50 kD, manufactured by MPBiomedicals), together with drinking water, was given to the mice forsix days.

Meanwhile, for preparing an oxazolone-induced colitis model, the micewere presensitized by transdermally applying, onto the mice, 150 μl of a3% oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one,Sigma-Aldrich)/100% ethanol solution. Five days after that, 150 μl of a1% oxazolone/50% ethanol solution was intrarectally administered againto the presensitized mice under alight anesthesia. Note that theintrarectal administration was conducted by using a 3.5 F catheter.

Each mouse was analyzed daily for body weight, occult blood, bleedingvisible with the naked eyes (gross blood), and the hardness of stool.Moreover, the body weight loss percentage, intestinal bleeding (nobleeding, occult blood (hemoccult+), or bleeding visible with the nakedeyes), and the hardness of stool (normal stool, loose stool, ordiarrhea) were evaluated numerically, and the disease activity index(DAI) was calculated in accordance with the description in “S. Wirtz, C.Neufert, B. Weigmann, M. F. Neurath, Nat Protoc 2, 541 (2007).”

<OVA Specific IgE Reaction>

BALB/c SPF mice were inoculated with a fecal suspension ofClostridium-colonized mice (2-week old), and grown in a conventionalenvironment. Then, 1 μg of OVA (grade V, Sigma) and 2 mg of alum (ThermoScientific), 0.2 ml in total, were intraperitoneally injected to themice (at their ages of 4 weeks and 6 weeks). Sera were collected everyweek from the mice at the root of their tail, and OVA-specific IgE wasmeasured by ELISA (Chondrex). Then, at their ages of 8 weeks, spleniccells were collected, inoculated in a 96-well plate at 1×10⁶ cells perwell, and stimulated with OVA (100 μg/ml) for three days. Thereafter,the culture supernatant was collected, and measured for IL-4 and IL-10levels by ELISA (R&D).

<Statistical Analysis>

The difference between control and experimental groups was evaluated bythe Student's t-test.

Example 1

First, it was investigated whether or not accumulation of regulatory Tcells (Treg cells) in the colonic lamina propria was dependent oncommensal bacteria. Specifically, lymphocytes were isolated fromperipheral lymph nodes (pLN) of Balb/c mice bred in the absence ofspecific pathogenic bacteria (SPF) or from lamina propria of the colonor the small intestine (SI) of the mice. The CD4 and Foxp3 were stainedby antibodies. Then, the ratio of Foxp3⁺ cells in CD4⁺ lymphocytes wasanalyzed by flow cytometry. FIG. 5 shows the obtained results. As isapparent from the results shown in FIG. 5, it was found that Foxp3⁺ Tregcells were present at a high frequency in the lamina propria of thegastrointestinal tracts, especially in the colonic lamina propria, ofthe mice kept under the environment free from specific pathogenicmicroorganisms (SPF). In addition, it was also found that the number ofthe Foxp3⁺ Treg cells in the colonic lamina propria gradually increasedup to three months after their birth, whereas the number of the Foxp3⁺Treg cells in the peripheral lymph nodes was basically constant from thetime of two weeks after their birth.

Example 2

Next, it was investigated whether or not the temporal accumulation ofthe Treg cells in the colon as found in Example 1 had a relationshipwith the colonization of intestinal commensal microbiota. Specifically,the expression of CD4 and the expression of Foxp3 in lymphocytesisolated from the small intestine, the colon, and the peripheral lymphnodes of mice bred under a germ-free (GF) or SPF environment (8 weeksold: Balb/c mice, IQI mice, and C57BL/6 mice) were analyzed. Similarresults were obtained in three or more independent experiments. FIGS. 6and 7 show the obtained results. Note that, in FIG. 7, each white circlerepresents the absolute number of CD4⁺ Foxp3⁺ cells in an individualmouse, and the error bars represent standard deviations (SDs).

In addition, lamina propria lymphocytes were collected from SPF mice andGF mice (Balb/c mice or C57BL/6 mice). CD4 and Foxp3 were stained withantibodies. Then, the lamina propria lymphocytes were analyzed by FACS.FIG. 8 shows the obtained results. Note that in FIG. 8 each white circlerepresents the absolute number of CD4⁺ Foxp3⁺ cells in an individualmouse, ** indicates that “P<0.001”, and * indicates that “P<0.01.”

Further, lymphocytes were isolated from the lamina propria of the colon,the lamina propria of the small intestine (SI), Peyer's patches (PPs),and mesenteric lymph nodes (MLNs) of mice (SPF C57BL/6 mice) to whichantibiotics were orally administered with water for eight weeks. CD4 andFoxp3 were stained with antibodies. Then, the lymphocytes were analyzedby FACS. Similar results were obtained in two or more independentexperiments. FIG. 9 shows the obtained results (the ratio of the Foxp3⁺cells in the CD4⁺ cells of an individual mouse). Note that the followingantibiotics were used in combination in accordance with the descriptionin the following document: ampicillin (A; 500 mg/L, Sigma) vancomycin(V; 500 mg/L, NACALAI TESQUE, INC.) metronidazole (M; 1 g/L, NACALAITESQUE, INC.) neomycin (N; 1 g/L, NACALAI TESQUE, INC.)

-   Rakoff-Nahoum, J. Paglino, F. Eslami-Varzaneh, S. Edberg, R.    Medzhitov, Cell 118, 229 (Jul. 23, 2004)-   Fagarasan et al., Science 298, 1424 (Nov. 15, 2002)

In FIG. 9, each white circle represents the absolute number of the CD4⁺Foxp3⁺ cells in an individual mouse, each horizontal bar represents theaverage value of the absolute numbers, * indicates that “P<0.01,” and“AVMN” represents the kinds of the administered antibiotics by using thefirst letters of the antibiotics.

As is apparent from the results shown in FIGS. 6 to 9, the frequenciesand the absolute numbers of Foxp3⁺ CD4⁺ cells in the small intestine andthe peripheral lymph nodes of the GF mice were equal to or greater thanthose of the SPF mice (refer to FIGS. 6 to 8). In addition, the numbersof the Treg cells in the small intestinal lamina propria, Peyer'spatches, and mesenteric lymph nodes of the SPF mice to which theantibiotics were orally administered for eight weeks were equal to orgreater than those of the SPF mice (refer to FIG. 9). Meanwhile, thenumber of the Foxp3⁺ CD4⁺ cells in the colonic lamina propria of the GFmice was decreased significantly in comparison with that of the SPF mice(refer to FIGS. 6 and 7). This decrease was commonly observed among miceof different genetic backgrounds (Balb/c, IQI, and C57BL/6), as well asamong mice bred in different animal facilities (refer to FIG. 7 for thedata regarding the different genetic backgrounds, the data regarding themice bred in the different animal facilities are not shown in thedrawings). In addition, it was also shown that the number of Treg cellsin the colonic lamina propria of the SPF C57BL/6 mice to which theantibiotics were administered was decreased significantly (refer to FIG.9).

Example 3

Next, it was directly checked whether or not the decrease in the numberof the Treg cells in the colonic lamina propria of the GF mice shown inExample 2 was attributed to the absence of microbiota. Specifically, afecal suspension of B6 SPF mice purchased from The Jackson Laboratorywas orally administered to GF-IQI mice (conventionalization). Threeweeks after the administration, lymphocytes were isolated from thecolonic lamina propria, and the expression of Foxp3 in CD4⁺ lymphocyteswas analyzed. FIGS. 10 and 11 show the obtained results. Note that eachwhite circle in FIG. 11 represents the absolute number of CD4⁺ Foxp3⁺cells in an individual mouse, the error bars represent standarddeviations (SD), * indicates that “P<0.01” in Student's t-test, and **indicates that “P<0.001.” As is apparent from the results shown in FIGS.10 and 11, the number of Treg cells in the small intestinal laminapropria did not change. However, the number of the Treg cells in thecolonic lamina propria increased significantly. Hence, it was shown thathost-microbial interaction played an important role in the accumulationof Foxp3⁺ Treg cells in the colonic lamina propria, while theaccumulation of the Treg cells in the small intestinal lamina propriahad a different mechanism.

Example 4

Next, the relationship between the gut-associated lymphoid tissues ofmice and the number of Foxp3⁺ cells in the colonic lamina propria of themice was investigated in accordance with the method described in M. N.Kweon et al., J Immunol 174, 4365 (Apr. 1, 2005). Specifically, 100 μgof an extracellular domain recombinant protein (a fusion protein(LTβR-Ig) between a lymphotoxin β receptor (LTβR) and a Fc region ofhuman IgG1, refer to Honda et al., J Exp Med 193, 621 (Mar. 5, 2001))was injected intraperitoneally into pregnant C57BL/6 mice 14 days afterconception. The LTβR-Ig was again injected intraperitoneally intofetuses obtained from such mice, so that mice from which isolatedlymphoid follicles (ILFs), Peyer's patches (PPs), and colonic-patches(CPs) were completely removed were produced. Then, the ratios of Foxp3⁺cells in CD4⁺ cells in the colonic lamina propria of the mice treatedwith the LTβR-Ig, and mice treated with rat IgG (control) were analyzedby FACS. FIG. 12 shows the obtained results. Note that in FIG. 12 eachwhite circle represents the ratio of Foxp3⁺ cells in an individualmouse, and the error bars represent standard deviations. As is apparentfrom the results shown in FIG. 12, it was found that the ratio of theFoxp3⁺ cells in the colonic lamina propria of the mice deficient inisolated lymphoid follicles, Peyer's patches, and the colonic-patches(the mice treated with the LTβR-Ig) rather increased. Accordingly, itwas suggested that the decrease in the number of the Treg cells in thecolonic lamina propria of the GF mice and the mice treated with theantibiotics was caused because the transmission of specific signalswhich promotes the accumulation of Treg cells in the colonic laminapropria and which is caused by the intestinal microbes did not occur,rather than simply because of a secondary effect of disorganizedgut-associated lymphoid tissues.

Example 5

To investigate whether or not a specific intestinal flora induced theaccumulation of colonic Treg cells, vancomycin as an antibiotic againstGram-positive bacteria or polymyxin B as an antibiotic againstGram-negative bacteria was administered to SPF mice (from 4 weeks ofage) for four weeks, and analyzed for the ratio of Foxp3⁺ cells in theCD4⁺ cell group ([%] Foxp3⁺ in CD4). FIG. 30 shows the obtained results.Note that, in FIG. 30, “SPF” indicates the result of SPF mice (control),“poly B” indicates the result of the SPF mice to which polymyxin B wasadministered, and “Vanco.” indicates the result of the SPF mice to whichvancomycin was administered. Meanwhile, * indicates that “P<0.01.”

As is apparent from the results shown in FIG. 30, the number of Tregcells in the colon of the mice to which vancomycin was administered wasmarkedly decreased in comparison with that of the control. In contrast,no influence was observed on the number of Treg cells of the mice towhich polymyxin B was administered. Those facts suggested thatGram-positive commensal bacteria played a major role in accumulation ofTreg cells.

Example 6

A recent report has suggested that spore-forming bacteria play animportant role in intestinal T cells response (see V. Gaboriau-Routhiauet al., Immunity 31, 677 (Oct. 16, 2009)). In this respect, fecalmicroorganisms (spore-forming fraction) resistant to 3% chloroform wereorally administered to GF mice, which were then analyzed for the ratioof Foxp3⁺ cells in the CD4⁺ cell group ([%] Foxp3⁺ in CD4). FIG. 31shows the obtained results. Note that, in FIG. 31, “GF” indicates theresult of GF mice, and “+chloro” indicates the result of the GF mice towhich the chloroform-treated feces were administered. Meanwhile, **indicates that “P<0.001.”

As is apparent from the results shown in FIG. 31, three weeks after theadministration of the chloroform-treated feces, the number of Treg cellsin the administered mice was markedly increased to the same level asthose of the SPF mice and the GF mice to which the untreated feces wasforcibly administered (see FIGS. 7 and 11).

Accordingly, considering the results shown in Example 5 in combination,it was revealed that the specific components of the indigenousmicrobiota were highly likely to belong to the Gram-positive group, andthat the spore-forming fraction played an important role in theinduction of Treg cells.

Example 7

Next, the species of the intestinal microbiota which induced theaccumulation of Treg cells in the colon as suggested in Examples 4 to 6were identified. Specifically, segmented filamentous bacteria (SFB), 16strains of the Bacteroides spp. (Bactero. (6 strains of B. vulgatus, 7of the B. acidifaciens group 1, and 3 of the B. acidifaciens group 2)),3 strains of the Lactobacillus (Lacto. (L. acidophilus, L. fermentum,and L. murinum)), and 46 strains of Clostridium spp. (Clost., refer to“Itoh, K., and Mitsuoka, T. Characterization of clostridia isolated fromfaeces of limited flora mice and their effect on caecal size whenassociated with germ-free mice. Lab. Animals 19: 111-118 (1985))”), ormicrobiota collected from mice (SPF) bred under a conventionalenvironment was orally administered to GF-Balb/c mice or GF-IQI mice.The mice were maintained in vinyl isolators for three weeks. Then, CD4cells were isolated from the colon and the small intestine of thesemice. The numbers of Treg cells in the colon and the small intestinewere analyzed by flow cytometry.

FIG. 13 shows FACS dot-plots obtained when a gate was set on CD4⁺ cellsof the Balb/c mice. FIG. 14 shows the ratio of Foxp3⁺ cells in CD4⁺cells of each mouse.

Note that, the bacteria belonging to the genus Clostridium areclassified by sequencing of 16S rRNA gene, as follows. Specifically, the16S rRNA genes of the bacteria were amplified by PCR using 16S rRNAgene-specific primer pairs: 5′-AGAGTTTGATCMTGGCTCAG-3′ (SEQ ID NO: 19)and 5′-ATTACCGCGGCKGCTG-3′ (SEQ ID NO: 20) (see T. Aebischer et al.,Vaccination prevents Helicobacter pylori-induced alterations of thegastric flora in mice. FEMS Immunol. Med. Microbiol. 46,221-229(2006)).The 1.5-kb PCR product was then introduced into pCR-Blunt Vector. Theinserts were sequenced and aligned using the ClustalW software program.The resulting sequences of 16S rRNA genes derived from strain 1-41 of 46strains of Clostridium spp. were shown in SEQ ID NO: 21-61. Phylogenetictree which was constructed by the neighbor-joining method with theresulting sequences of the 41 strains of Clostridium and those of knownbacteria obtained from Genbank database using Mega software was shown inFIG. 49.

As is apparent from the results shown in FIGS. 13 and 14, no effect onthe number of the Treg cells in the colon was observed in the GF mice inwhich the segmented filamentous bacteria (SFB) were colonized (refer toFIG. 14). Moreover, mice in which the cocktail of three strains ofLactobacillus was colonized gave similar results (refer to FIG. 14). Onthe other hand, it was shown that the accumulation of Foxp3⁺ cells inthe colonic lamina propria was strongly induced in the mice in which 46strains of Clostridium spp. were colonized. Importantly, suchaccumulation was promoted irrespective of the genetic backgrounds of themice, and led to the increase in number similar to that in the SPF micealthough intestinal microbiota of only a single genus were colonized. Itwas also shown that the colonization of the Clostridium did not changethe number of Treg cells in the small intestinal lamina propria (referto FIG. 14). Note that, when the 16 strains of Bactericides spp. werecolonized, the number of Treg cells in the colon was increasedsignificantly. However, the extent of the increase varied depending onthe genetic background of the mice in which the bacteria were colonized(refer to FIGS. 13 and 14).

Example 8

Next, CD4 expression, Foxp3 expression, and Helios expression in LPlymphocytes of the thymuses and the colons of SPF mice, GF mice,Lactobacillus-colonized mice, and Clostridium-colonized mice wereanalyzed by flow cytometry.

FIGS. 32 and 33 show the obtained results. Note that, in FIGS. 32 and33, “GF” or “Germ. Free” indicates the results of the GF mice, “SPF”indicates the results of the SPF mice, “Lacto.” indicates the results ofthe Lactobacillus-colonized mice, and “Clost.” indicates the results ofthe Clostridium-colonized mice. In FIG. 32, the vertical axis representsthe ratio of Helios cells in the Foxp3⁺ cell group ([%] Helios⁻ inFoxp3⁺), and ** indicates that “P<0.001.”

As is apparent from the results shown in FIGS. 32 and 33, most Foxp3⁺cells found in the SPF mice or the Clostridium-colonized mice did notexpress Helios. Note that Helios is a transcription factor known to beexpressed in thymic-derived natural Treg cells (see A. M. Thornton etal., J Immunol 184, 3433 (Apr. 1, 2010)). Accordingly, it was suggestedthat most of the Treg cells in the SPF mice and theClostridium-colonized mice were Treg cells induced in peripheralportions, i.e., so-called iTreg cells.

Example 9

Next, it was investigated whether or not the colonization of theClostridium or the like had an influence on other T cells. Specifically,SFB, 16 strains of Bacteroides spp. (Bactero.), 46 strains ofClostridium spp. (Clost.), or microbiota collected from mice bred undera conventional environment (SPF) was colonized in GF IQI mice. Threeweeks later, lymphocytes in the colonic lamina propria were isolatedfrom these mice, and stimulated with PMA (50 ng/ml) and ionomycin (1μg/ml) for four hours in the presence of Golgistop (BD Bioscience).After the stimulation was given, intracellular cytokines were stained byusing an anti-IL-17 PE antibody (TC11-18H10) and an anti-IFN-g FITCantibody (BD Bioscience) in accordance with the manual of acytofix/cytoperm kit (BD Bioscience). Then, the ratio of IFN-γ⁺ cells orIL-17⁺ cells in CD4⁺ leucocytes was analyzed by flow cytometry. FIGS. 15and 16 show the obtained results. Note that, in FIGS. 15 and 16, eachwhite circle represents the absolute number of CD4⁺ IFN-γ⁺ cells or theabsolute number of CD4⁺ IL-17⁺ cells in each individual mouse, and theerror bars represent standard deviations (SD). As is apparent from theresults shown in FIGS. 15 and 16, the colonization of the Clostridiumdid not have any influence on Th1 cells (CD4⁺ IFN-γ⁺ cells) in thecolon, and caused only a slight increase of Th17 cells (CD4⁺ IL-17⁺cells). Accordingly, it was suggested that the genus Clostridium was agenus of bacteria which specifically induced Treg cells.

Example 10

It has been reported that 46 strains of Clostridium spp. exert aninfluence on the accumulation of CD8⁺ intestinal tract intraepitheliallymphocytes (IELs) in the colon. Accordingly, it is conceivable thatClostridium regulates the immune system in various aspects, and thatClostridium exhibits a marked ability to induce and maintain Treg cellsespecially in the colon, as described above. In addition, a kind ofcytokines, transforming growth factor-β (TGF-β), is known to play animportant role in regulation of Treg cell generation.

In this respect, it was examined whether or not the colonization ofClostridium provided a colonic environment rich in TGF-β. Specifically,first, the whole colons of GF mice, Clostridium-colonized mice, andLactobacillus-colonized mice were cultured for 24 hours, and the culturesupernatants thereof were measured for the concentration of active TGF-β(TGF-β1) by ELISA (the number of mice analyzed was four per group). FIG.34 shows the obtained results. Note that, in FIG. 34, “GF” indicates theresult of the GF mice, “Clost.” indicates the result of theClostridium-colonized mice, and “Lacto.” indicates the result ofLactobacillus-colonized mice. Meanwhile, * indicates that “P<0.02,” and** indicates that “P<0.001.”

As is apparent from the results shown in FIG. 34, the amount of TGF-βproduced in the colons of the Clostridium-colonized mice wassignificantly larger than those of the GF mice and theLactobacillus-colonized mice.

Next, intestinal epithelial cells (IECs) of GF mice andClostridium-colonized mice were cultured for 24 hours, and the culturesupernatants thereof were measured for the concentration of active TGF-β(TGF-β1) by ELISA (the number of mice analyzed was four per group). FIG.35 shows the obtained results. Note that, in FIG. 35, “GF” indicates theresult of the GF mice, and “Clost.” indicates the result of theClostridium-colonized mice. Meanwhile, ** indicates that “P<0.001.”

As is apparent from the results shown in FIG. 35, TGF-β was detected inthe culture supernatant of the IECs isolated from theClostridium-colonized mice, whereas no TGF-β was detected in the culturesupernatant of the IECs isolated from the GF mice.

Next, as described above, splenic CD4⁺ T cells were cultured for fivedays together with a 50% conditioned medium in which IECs isolated fromthe GF mice or the Clostridium-colonized mice were cultured, and withthe anti-CD3 antibody, in the presence or absence of an anti-TGF-βantibody. Then, the T cells were collected, and analyzed for expressionof Foxp3 by real-time RT-PCR. FIG. 36 shows the obtained results. Notethat, in FIG. 36, “Medium” indicates the result of a medium in which nocells were cultured, “GF” indicates the result of the conditioned mediumin which the IECs of the GF mice were cultured, “Clost.” indicates theresult of the conditioned medium in which the IECs of theClostridium-colonized mice were cultured, and “Clost.+αTGFβ” indicatesthe result of the conditioned medium to which the anti-TGF-β antibodywas added and in which the IECs of the Clostridium-colonized mice werecultured. Meanwhile, ** indicates that “P<0.001.”

As is apparent from the results shown in FIG. 36, when the culturesupernatant of the IECs derived from the Clostridium-colonized mice wasadded to the splenic CD4⁺ T cells, the differentiation intoFoxp3-expressing cells was accelerated. Meanwhile, the differentiationinto the Treg cells was inhibited by the anti-TGF-β antibody.

Moreover, the expression of MMP2, MMP9, and MMP13, which are thought tocontribute to the activation of latent TGF-β was investigated. Theexpression of indoleamine 2,3-dioxygenase (IDO), which is thought to beinvolved in the induction of Treg cells, was also investigated.Specifically, 46 bacterial strains of the genus Clostridium (Clost.), orthree bacterial strains of the genus Lactobacillus (Lacto.) were orallyadministered to C57BL/6 germ-free mice. Three weeks after theadministration, IECs were collected, and analyzed for relative mRNAexpression levels of MMP2, MMP9, MMP13, and IDO genes by real-timeRT-PCR (the number of mice analyzed was three per group). FIGS. 37 to 40show the obtained results. Note that, in FIGS. 37 to 40, “GF#1 to 3”indicate the results of GF mice, “Clost.#1 to 3” indicate the results ofthe Clostridium-colonized mice, and “Lacto.#1 to 3” indicate the resultsof the Lactobacillus-colonized mice.

For the relationship between the activation of latent TGF-β and theabove-describe MMP, see D'Angelo et al., J. Biol. Chem. 276,11347-11353, 2001; Heidinger et al., Biol. Chem. 387, 69-78, 2006; Yu etal., Genes Dev. i4, 163-176, 2000. For the relationship between IDO andthe induction of Treg cells, see G. Matteoli et al., Gut 59, 595 (May,2010).

As is apparent from the results shown in FIGS. 37 to 39, in agreementwith the production of TGF-β described above, transcription products ofthe genes encoding MMP2, MMP9, and MMP13 were expressed at higher levelsin the IECs derived from the Clostridium-colonized mice than those inthe GF mice and in the Lactobacillus-colonized mice.

Moreover, as is apparent from the results shown in FIG. 40, IDO wasexpressed only in the Clostridium-colonized mice.

Accordingly, it was revealed that the Clostridium activated the IECs,and led to the production of TGF-β and other Treg cell-inducingmolecules in the colon.

Example 11

Next, it was investigated whether or not the Treg cell accumulationinduced by the colonization of the Clostridium was dependant on signaltransmission by pathogen-associated molecular pattern recognitionreceptors. Specifically, the numbers of Treg cells in the colonic laminapropria of each of SPF mice of Myd88 (deficient in Myd88 (signalingadaptor for Toll-like receptor)), Rip2^(−/−) (deficient in Rip2 (NODreceptor adaptor)), and Card9^(−/−) (deficient in Card9 (essentialsignal transmission factor for Dectin-1 signal transmission)) wereexamined. In addition, Clostridium spp. were caused to be colonized inthe Myd88^(−/−) GF mice, and the change in the number of Treg cells wasinvestigated. FIGS. 17 and 18 show the obtained results. As is apparentfrom the results shown in FIGS. 17 and 18, the number of Treg cells ofeach kind of the SPF mice deficient in the associated factors of thepathogen-associated molecular pattern recognition receptors did notchange relative to that of wild-type mice of the same litter, whichserved as a control. In addition, it was found that also whenClostridium spp. were colonized in GF mice deficient in Myd88, theaccumulation of Treg cells in the colonic lamina propria was induced.Accordingly, it has been suggested that the mechanism of inducing theaccumulation of Treg cells in the colonic lamina propria relies not onactivation of recognition pathway for major pathogen-associatedmolecular patterns as is caused by most of bacterium, but on specificcommensal bacterial species.

Example 12

Intestinal tract Foxp3⁺ Treg cells are known to exert someimmunosuppressive functions through IL-10 production (refer toNon-Patent Document 9). Meanwhile, animals having CD4⁺ Foxp3⁺ cells fromwhich IL-10 is specifically removed are known to develop inflammatorybowel disease (refer to Non-Patent Document 18). In this respect, first,the expression of IL-10 in lymphocytes of various tissues was examined.Specifically, lymphocytes were isolated from various tissues of SPFIl10^(venus) mice, and the expression of CD4 and the expression of Venuswere analyzed by flow cytometry. FIG. 19 shows the obtained results.Note that each numeric value in FIG. 19 represents the ratio of cellswithin the corresponding one of regions divided into four.

Moreover, lymphocytes in the colonic lamina propria were isolated fromIl10^(venus) mice, and the expression of T cell receptor β chain (TCRβ)on the surfaces of the cells was detected by FACS. FIG. 20 shows theobtained results (FACS dot-plots obtained when a gate was set on CD4⁺cells). Note that each numeric value in FIG. 20 represents the ratio ofcells within the corresponding one of regions divided into four.

Furthermore, lymphocytes in the colonic lamina propria were isolatedfrom Il10^(venus) mice. The lymphocytes were stimulated with PMA (50ng/ml) and ionomycin (1 μg/ml) for four hours in the presence ofGolgistop (BD Bioscience). Then, after the stimulation was given,intracellular cytokines were stained by using an anti-IL-17 PE antibody,an anti-IL-4 APC antibody (11B11), and an anti-IFN-g FITC antibody (BDBioscience) in accordance with the manual of a cytofix/cytoperm kit (BDBioscience). FIG. 21 shows the obtained results (FACS dot-plots obtainedwhen a gate was set on CD4⁺ cells).

Note that each numeric value in FIG. 21 represents the ratio of cellswithin the corresponding one of regions divided into four.

In addition, Foxp3⁺ CD4⁺ cells and Foxp3⁻ CD4⁺ cells were isolated fromthe spleen (Spl) of Foxp3^(eGFP) reporter mice, and Venus⁺ cells wereisolated from the colonic lamina propria and the small intestine (SI)lamina propria of Il10^(venus) mice. Then, the obtained cells wereanalyzed in terms of the expression of predetermined genes. The geneexpression was analyzed by real-time RT-PCR using a Power SYBR Green PCRMaster Mix (Applied Biosystems) and an ABI 7300 real time PCR system(Applied Biosystems). Here, the value for each cell was normalized forthe amount of GAPDH. FIG. 22 shows the obtained results. Note that inFIG. 22 the error bars represent standard deviations.

As is apparent from the results shown in FIGS. 19 to 22, almost noVenus⁺ cells (IL-10-producing cells) were detected in the cervical lymphnodes (peripheral lymph nodes), thymus, peripheral blood, lung, andliver of mice kept under the SPF conditions. Meanwhile, in the spleen,Peyer's patches, and mesenteric lymph nodes thereof, Venus⁺ cells wereslightly detected (refer to FIG. 19). On the other hand, many Venus⁺cells were found in the lymphocytes in the small intestine laminapropria and colonic lamina propria. In addition, most of the Venus⁺cells in the intestines were positive for CD4, and also positive for Tcell receptor β chain (TCRβ) (refer to FIGS. 19 and 20). Moreover, itwas found that the Venus⁺ CD4⁺ T cells expressed Foxp3 and other Tregcell-associated factors such as a cytotoxic T-Lymphocyte antigen(CTLA-4) and a glucocorticoid-induced TNFR-associated protein (GITR)although the Venus⁺ CD4⁺ T cells showed none of the phenotypes of Th2(IL-4-producing) and Th17 (IL-17-producing) (refer to FIGS. 21 and 22).In addition, it was shown that the expression level of CTLA-4 in theintestinal Venus⁺ cells was higher than that in the splenic GFP⁺ Tregcells isolated from the Foxp3^(eGFP) reporter mice (refer to FIG. 22).

Example 13

Venus⁺ cells can be classified into at least two subsets, namely, Venus⁺Foxp3⁺ double positive (DP) Treg cells and Venus⁺ Foxp3⁻ Treg cells onthe basis of intracellular Foxp3 expression. Cells of the latter subsetcorrespond to type 1 regulatory T cells (Tr1) (refer to Non-PatentDocuments 8 and 9). In this respect, the Venus⁺ cells (IL-10-producingcells) observed in Example 8 were investigated in terms of theexpression of Foxp3. Specifically, the expression of CD4, Foxp3, andVenus in the lamina propria of the colon and the lamina propria of thesmall intestine of Il10^(venus) mice kept under GF or SPF conditions wasanalyzed by FACS, and the numbers of Venus⁺ cells in the intestinaltract lamina propria were compared between SPF and GF Il10^(venus) mice.FIG. 23 shows the obtained results (dot-plots obtained when a gate wasset on CD4⁺ cells).

In addition, the intracellular expression of Venus and Foxp3 in CD4cells in various tissues of SPF Il10^(venus) mice was analyzed by flowcytometry. FIG. 24 shows the obtained results (dot-plots obtained when agate was set on CD4⁺ cells). Note that each numeric value in FIG. 24represents the ratio of cells within the corresponding one of regionsdivided into four.

Moreover, in order to investigate whether or not the presence ofcommensal bacteria had any influence on the expression of IL-10 inregulatory cells in the gastrointestinal tracts, germ-free (GF)Il10^(venus) mice were prepared. Then, predetermined species of bacteriawere caused to be colonized in the obtained GF Il10^(venus) mice. Threeweeks after the species of bacteria were colonized, a CD4⁺ cell group(V⁺F⁻, Venus⁺ Foxp3⁻ cells; V⁺F⁺, Venus⁺ Foxp3⁺ cells; and V⁻F⁺, Venus⁻Foxp3⁺ cells) in which Foxp3 and/or Venus were expressed in the colonand the small intestine was analyzed by flow cytometry. FIG. 25 showsdot-plots obtained when a gate was set on colonic CD4⁺ cells, and FIGS.26 and 27 show the ratios in the CD4⁺ cell group of each mouse. Notethat each numeric value in FIG. 25 represents the ratio of cells withinthe corresponding one of regions divided into four. Meanwhile, the errorbars in FIGS. 26 and 27 represent standard deviations, * indicates that“P<0.02,” and ** indicates that “P<0.001.”

Moreover, in order to check whether or not the presence of commensalbacteria had any influence on the expression of IL-10 in regulatorycells in the gastrointestinal tracts, antibiotics were orally given withwater to five or six Il10^(venus) mice per group for 10 weeks. Thefollowing antibiotics were used in combination.

ampicillin (A; 500 mg/L Sigma)vancomycin (V; 500 mg/L NACALAI TESQUE, INC.)metronidazole (M; 1 g/L NACALAI TESQUE, INC.)neomycin (N; 1 g/L NACALAI TESQUE, INC.)

Then, CD4 and Foxp3 of lymphocytes in the lamina propria of the colon,the lamina propria of the small intestine (SI), mesenteric lymph nodes(MLN), and Peyer's patches (PPs) were stained with antibodies, andanalyzed by FACS. The results were obtained from two or more independentexperiments which gave similar results. FIG. 28 shows the obtainedresults (the ratio of Venus⁺ cells in CD4⁺ cells in each sample). Notethat each white circle in FIG. 28 represents an individual sample, eachhorizontal bar represents an average value, * indicates that “P<0.02,”and “AVMN” represents the kinds of the administered antibiotics by usingthe first letters of the antibiotics.

As is apparent from the results shown in FIGS. 23 and 24, it was shownthat the small intestinal lamina propria was rich in Venus⁺ Foxp3 cells,namely, Tr1-like cells, and that the Venus⁺ Foxp3⁺ DP Treg cells werepresent at a high frequency in the colon of the SPF mice (refer to FIGS.23 and 24). In contrast, although sufficient numbers of Foxp3⁺ cellswere observed also in other tissues, the expression of Venus was notobserved in almost all of the cells (refer to FIG. 24).

In addition, as is apparent from the results shown in FIGS. 23 and 25 to28, it was shown that all regulatory T cell fractions of Venus⁺ Foxp3⁻,Venus⁺ Foxp3⁺, and Venus⁻ Foxp3⁺ in the colon significantly decreasedunder the GF conditions (FIGS. 23 and 26 to 27). Moreover, similardecrease in Venus⁺ cells was observed also in the SPF Il10^(venus) micetreated with the antibiotics (refer to FIG. 28).

Moreover, as is apparent from the results shown in FIGS. 25 to 27, thecolonization of Clostridium spp. strongly induced all regulatory T cellfractions of Venus⁺ Foxp3⁻, Venus⁺ Foxp3⁺, and Venus⁻ Foxp3⁺ in thecolon, and the degrees of the induction thereof were equal to those inthe SPF mice (refer to FIGS. 25 and 27). In addition, it was found thatthe colonization of the three strains of Lactobacillus or thecolonization of SFB had an extremely small influence on the number ofVenus⁺ and/or Foxp3⁺ cells in the colon (refer to FIGS. 25 and 27).Moreover, the colonization of 16 strains of Bacteroides spp. alsoinduced Venus⁺ cells, but the influence of the colonization was specificto Venus⁺ Foxp3Tr1-like cells (refer to FIGS. 25 and 27). On the otherhand, it was found that none of the bacterial species tested exerted anysignificant influence on the number of IL-10-producing cells in thesmall intestinal lamina propria (refer to FIG. 26).

Hence, it was shown that the genus Clostridium colonized in the colon ora physiologically active substance derived from the bacteria provided asignal for inducing the accumulation of IL-10⁺ regulatory T cells in thecolonic lamina propria or the expression of IL-10 in T cells. Meanwhile,it was shown that the number of Venus⁺ cells in the small intestine wasnot significantly influenced by the situation where no commensalbacteria were present or commensal bacteria were decreased (refer toFIGS. 23 and 26 to 28), and that IL-10⁺ regulatory cells (Tr1-likecells) accumulated in the small intestinal lamina propria independentlyof commensal bacteria.

Example 14

It was investigated whether or not Venus⁺ cells induced by the genusClostridium had an immunosuppressive function similar to that of Venus⁺cells in the colon of SPF mice. Specifically, CD4⁺ CD25⁻ cells (effectorT cells, Teff cells) isolated from the spleen were seeded in aflat-bottomed 96-well plate at 2×10⁴/well, and cultured for three daystogether with 2×10⁴ splenic CD11c⁺ cells (antigen-representing cells)subjected to 30 Gy radiation irradiation treatment, 0.5 μg/ml of ananti-CD3 antibody, and a lot of Treg cells. In addition, for the lastsix hours, the CD4⁺ CD25 cells were cultured, with [³H]-thymidine (1μCi/well) was added thereto. Note that, Treg cells used in Example 14were CD4⁺ GFP⁺ T cells isolated from the spleen of Foxp3^(eGFP) reportermice, or CD4⁺ Venus⁺ T cells in the colonic lamina propria of GFIl10^(venus) mice in which Clostridium spp. were colonized or SPFIl10^(venus) mice. Then, proliferation of the cells was determined basedon the uptake amount of [³H]-thymidine, and represented by a count perminute (cpm) value.

As is apparent from the results shown in FIG. 29, Venus⁺ CD4⁺ cells ofthe mice in which the genus Clostridium was colonized suppressed invitro proliferation of CD25⁻ CD4⁺ activated T cells. The suppressionactivity was slightly inferior to that of GFP⁺ cells isolated from theFoxp3^(eGFP) reporter mice, but equal to that of Venus⁺ cells isolatedfrom the SPF Il10^(Venus) mice. Accordingly, it has been shown that thegenus Clostridium induces IL-10-expressing T cells having sufficientimmunosuppressive activities, and thereby plays a critical role inmaintaining immune homeostasis in the colon.

Example 15

Next, the influence, on the local immune response, of the colonizationof a large number of Clostridium and the resultant proliferation of Tregcells was investigated.

<Dextran Sulfate Sodium (DSS)-Induced Colitis Model>

First, the DSS-induced colitis model was prepared as described above,and the influence, on the model mice, of the inoculation of theClostridium and the proliferation of Treg cells was investigated.Specifically, control mice and Clostridium-inoculated mice were treatedwith 2% DSS, then observed and measured for six days for the body weightloss, the hardness of stool, and bleeding, and then were evaluatednumerically. In addition, on day 6, the colons were collected,dissected, and analyzed histologically by HE staining. FIGS. 41 to 43show the obtained results. Note that, in FIGS. 41 to 43, “SPF+Clost.” or“SPF+Clost.#1 to 3” indicate the results of C57BL/6 mice inoculated witha fecal suspension of Clostridium-colonized mice, and grown in aconventional environment for six weeks, and “SPF” or “SPF#1 to 3”indicate the results of C57BL/6 mice (control mice) grown in aconventional environment for six weeks without being inoculated with thefecal suspension. In addition, in FIG. 41, the vertical axis “Diseasescore” represents the disease activity index (DAI) described above, andthe horizontal axis “post 2% DSS(d)” represents the days elapsed afterthe initial administration of 2% DSS to the mice. Moreover, in FIG.41, * indicates that “P<0.02,” and ** indicates that “P<0.001.”Meanwhile, Treg cells induced by regulatory dendritic cells are known toplay a preventive role in a DSS-induced colitis model (see S.Manicassamy et al., Science 329, 849 (Aug. 13, 2010)).

As is apparent from the results shown in FIGS. 41 to 43, the symptoms ofthe colitis such as body weight loss and rectal bleeding weresignificantly suppressed in the mice having a large number ofClostridium (hereinafter also referred to as “Clostridium-abundantmice”) in comparison with the control mice (see FIG. 41). All thefeatures typical for colonic inflammation, such as shortening of thecolon, edema, and hemorrhage, were observed markedly in the control micein comparison with the Clostridium-abundant mice (see FIG. 42).Moreover, histological features such as mucosal erosion, edema, cellularinfiltration, and crypt loss were less severe in the DSS-treatedClostridium-abundant mice than in the control mice (see FIG. 43).

<Oxazolone-Induced Colitis Model>

Next, the oxazolone-induced colitis model was prepared as describedabove, and the influence, on the model mice, of the inoculation ofClostridium and the proliferation of Treg cells was investigated.Specifically, control mice and Clostridium-inoculated mice weresensitized with oxazolone, and subsequently the inside of the rectumsthereof were treated with a 1% oxazolone/50% ethanol solution. Then, thebody weight loss was observed and measured. In addition, the colons weredissected, and analyzed histologically by HE staining. FIGS. 44 and 45show the obtained results. Note that, in FIGS. 44 and 45, “SPF+Clost.”indicates the results of C57BL/6 mice (Clostridium-abundant mice)inoculated with a fecal suspension of Clostridium-colonized mice, andgrown in a conventional environment for six weeks, and “SPF” indicatesthe results of C57BL/6 mice (control mice) grown in a conventionalenvironment for six weeks without being inoculated with the fecalsuspension. In addition, in FIG. 44, the vertical axis “Weight (% ofinitial)” represents the body weight after the administration of 1%oxazolone where the body weight before the administration was taken as100%, and the horizontal axis “post 1% oxazolone (d)” represents thedays elapsed after the administration of 1% oxazolone to the mice.Meanwhile, it is known that Th2-type T cells are involved in colitisinduced by oxazolone. (see M. Boirivant, I. J. Fuss, A. Chu, W. Strober,J Exp Med 188, 1929 (Nov. 16, 1998)).

As is apparent from the results shown in FIGS. 44 and 45, the colitisproceeded along with persistent body weight loss in the control mice.Meanwhile, the body weight loss of the Clostridium-abundant mice wasreduced (see FIG. 44). In addition, it was also revealed that portionshaving histological diseases such as mucosal erosion, edema, cellularinfiltration, and hemorrhage were reduced in the colon of theClostridium-abundant mice (see FIG. 45).

Example 16

Next, the influence, on the systemic immune response (systemic IgEproduction), of the colonization of a large number of Clostridium andthe resultant proliferation of Treg cells was investigated.Specifically, as described above, control mice andClostridium-inoculated mice were immunized by administeringalum-absorbed ovalbumin (OVA) twice at a 2-week interval. Then, serawere collected from these mice, and the OVA-specific IgE level thereofwas investigated by ELISA. In addition, splenic cells were collectedfrom the mice in each group, and IL-4 and IL-10 production by in-vitroOVA restimulation was investigated. FIGS. 46 to 48 show the obtainedresults. Note that, in FIGS. 46 to 48, “SPF+Clost.” indicates theresults of BALB/c SPF mice (Clostridium-abundant mice) inoculated with afecal suspension of Clostridium-colonized mice, and grown in aconventional environment, “SPF” indicates the results of BALB/c SPF mice(control mice) grown in a conventional environment without beinginoculated with the fecal suspension, and ** indicates that “P<0.001.”Meanwhile, in FIG. 46, the vertical axis “OVA-specific IgE (ng/ml)”represents the concentration of OVA-specific IgE in the sera. Moreover,in FIG. 46, the horizontal axis represents the days elapsed after theinitial administration of the alum-absorbed ovalbumin to theClostridium-abundant mice or the control mice (4-week old), and“OVA+Alum” indicates the timing of the administration of thealum-absorbed ovalbumin. In addition, in FIGS. 47 and 48, “OVA” on thehorizontal axis indicates the results in the case where the in-vitro OVArestimulation was performed, and “-” indicates the results in the casewhere no in-vitro OVA restimulation was performed. Moreover, in FIGS. 47and 48, the vertical axes “IL-4 (pg/ml)” and “IL-10 (pg/ml)” show theIL-4 concentration and the IL-10 concentration in culture supernatantsof splenic cells, respectively.

As is apparent from the results shown in FIGS. 46 to 48, the IgE levelwas significantly lower in the Clostridium-abundant mice than in thecontrol mice (see FIG. 46). Moreover, the IL-4 production by the OVArestimulation was reduced (see FIG. 47) and the IL-10 production therebywas increased (see FIG. 48) in the splenic cells of theClostridium-abundant mice sensitized with OVA and alum, in comparisonwith those of the control mice.

Accordingly, in consideration of the results shown in Example 15 incombination, it has been revealed that the induction of Treg cells byClostridium in the colon plays an important role in local and systemicimmune responses.

Example 17

Next, GF Balb/c were colonized with three strains of Clostridiumbelonging to cluster IV (strains 22, 23 and 32 listed in FIG. 49). Threeweeks later, colonic Foxp3⁺ Treg cells were analyzed by FACS. FIG. 50shows the obtained results. As is apparent from the results shown inFIG. 50, gnotobiotic mice colonized with three strains of Clostridiumshowed an intermediate pattern of Treg induction between GF mice andmice inoculated with all 46 strains.

Example 18

Next, it was investigated whether or not a spore-forming (for example, achloroform resistant) fraction of a fecal sample obtained from humanshad the effect of inducing proliferation or accumulation of regulatory Tcells similar to the spore-forming fraction of the fecal sample obtainedfrom mice.

Specifically, human stool from a healthy volunteer (Japanese, male, 29years old) was suspended with phosphate-buffered saline (PBS), mixedwith chloroform (final concentration 3%), and then incubated in ashaking water bath for 60 min. After evaporation of chloroform bybubbling with N₂ gas, the aliquots containing chloroform-resistant (forexample, spore-forming) fraction of human intestinal bacteria wereorally inoculated into germ-free (GF) mice (IQI, 8 weeks old). Thetreated mice were kept in a vinyl isolator for 3 weeks. The colon wascollected and opened longitudinally, washed to remove fecal content, andshaken in Hanks' balanced salt solution (HBSS) containing 5 mM EDTA for20 min at 37° C. After removing epithelial cells and fat tissue, thecolon was cut into small pieces and incubated with RPMI1640 containing4% fetal bovine serum, 1 mg/ml collagenase D, 0.5 mg/ml dispase and 40μg/ml DNase I (all manufactured by Roche Diagnostics) for 1 hour at 37°C. in a shaking water bath. The digested tissue was washed with HBSScontaining 5 mM EDTA, resuspended in 5 ml of 40% Percoll (manufacturedby GE Healthcare) and overlaid on 2.5 ml of 80% Percoll in a 15-mlFalcon tube. Percoll gradient separation was performed by centrifugationat 780 g for 20 min at 25° C. The interface cells were collected andsuspended in staining buffer containing PBS, 2% FBS, 2 mM EDTA and 0.09%NaN₃ and stained for surface CD4 with Phycoerythrin-labeled anti-CD4Ab(RM4-5, manufactured by BD Biosciences). Intracellular staining of Foxp3was performed using the Alexa647-labeled anti-Foxp3 Ab (FJK-16 s,manufactured by eBioscience) and Foxp3 Staining Buffer Set (manufacturedby eBioscience). The percentage of Foxp3 positive cells within the CD4positive lymphocyte population was analyzed by flow cytometry. FIGS. 51and 52 show the obtained results.

In figures, representative histograms (FIG. 51) and combined data (FIG.52) for Foxp3 expression by CD4 positive lymphocytes from GF mice (GF)or GF mice gavaged with chloroform-treated human stool (GF+Chloro.) areshown. In addition, numbers in FIG. 51 indicate the percentages of cellsin the gate. Each circle in FIG. 52 represents a separate animal, errorbars indicate the SD, and ** indicates that “P<0.001.”

As is apparent from the results shown in FIGS. 51 and 52, it was foundthat also when the spore-forming (for example, the chloroform resistant)fraction of human intestinal bacteria was colonized in GF mice, theaccumulation of Foxp3⁺ regulatory (Treg) cells in the colonic laminapropria of the mice was induced.

Next, it was investigated what species of bacteria grew by gavaging withchloroform-treated human stool.

Specifically, using a QIAamp DNA Stool mini kit (manufactured byQIAGEN), bacterial genomic DNA was isolated from the human stool from ahealthy volunteer as described above (human stool) or fecal pellets fromGF mice gavaged with chloroform-treated human stool (GF+Chloro.).Quantitative PCR analysis was carried out using a LightCycler 480(manufactured by Roche). Relative quantity was calculated by the ΔCtmethod and normalized to the amount of total bacteria, dilution, andweight of the sample. The following primer sets were used:

total bacteria (SEQ ID NO: 62) 5′-GGTGAATACGTTCCCGG-3′ and(SEQ ID NO: 63) 5′-TACGGCTACCTTGTTACGACTT-3′Clostridium cluster XIVa (Clostridium coccoides subgroup)(SEQ ID NO: 64) 5′-AAATGACGGTACCTGACTAA-3′ and (SEQ ID NO: 65)5′-CTTTGAGTTTCATTCTTGCGAA-3′ Clostridium cluster IV (Clostridium leptum)(SEQ ID NO: 66) 5′-GCACAAGCAGTGGAGT-3′ and (SEQ ID NO: 24)5′-CTTCCTCCGTTTTGTCAA-3′ Bacteroides (SEQ ID NO: 67)5′-GAGAGGAAGGTCCCCCAC-3′ and (SEQ ID NO: 68) 5′-CGCTACTTGGCTGGTTCAG-3′.

FIG. 53 shows the obtained results.

As is apparent from the results shown in FIG. 53, mice gavaged withchloroform-treated human stool exhibited high amounts of spore-formingbacteria, such as Clostridium clusters XIVa and IV, and a severedecrease of non-spore-forming bacteria, such as Bacteroides, comparedwith the human stool before chloroform treatment.

INDUSTRIAL APPLICABILITY

As has been described above, the present invention makes it possible toprovide an excellent composition for inducing proliferation oraccumulation of regulatory T cells (Treg cells) by utilizing bacteriabelonging to the genus Clostridium or a physiologically active substanceor the like derived from the bacteria. Since the composition of thepresent invention has immunosuppressive effects, the composition can beused, for example, to prevent or treat autoimmune diseases or allergicdiseases, as well as to suppress immunological rejection in organtransplantation or the like. In addition, healthy individuals can easilyand routinely ingest the composition as a food or beverage, such as ahealth food, to improve their immune functions.

SEQUENCE LISTING SEQ ID NO:1 to 20, 62 to 69

<223> Artificially synthesized primer sequence

SEQ ID NO:21 to 61

<223>16S rRNA coding gene sequence of each Clostridium strain

1. A method of treating autoimmune disease in a subject, the methodcomprising administering a pharmaceutical composition comprising apurified bacterial mixture of at least two live bacterial strainsbelonging to Clostridium clusters IV and XIVa to the subject in anamount sufficient to treat autoimmune disease.
 2. The method of claim 1,wherein the autoimmune disease is organ transplant rejection,inflammatory bowel disease (IBD), ulcerative colitis, Crohn's disease,sprue, rheumatoid arthritis, Type 1 diabetes, graft versus host disease,or multiple sclerosis.
 3. The method of claim 2, wherein the autoimmunedisease is inflammatory bowel disease (IBD), ulcerative colitis, orCrohn's disease.
 4. The method of claim 1, wherein the two livebacterial strains belonging to Clostridium clusters IV and XIVa compriseone or more strains belonging to Clostridium cluster IV.
 5. The methodof claim 1, wherein the two live bacterial strains belonging toClostridium clusters IV and XIVa comprise one or more strains belongingto Clostridium cluster XIVa.
 6. The method of claim 1, wherein the twolive bacterial strains belonging to Clostridium clusters IV and XIVacomprise one or more strains belonging to Clostridium cluster IV and oneor more strains belonging to Clostridium cluster XIVa.
 7. The method ofclaim 1, wherein the bacteria are human-derived bacteria.
 8. The methodof claim 1, wherein the bacteria are isolated from a chloroform-treatedfecal sample.
 9. The method of claim 1, wherein the bacteria arespore-forming bacteria.
 10. The method of claim 1, wherein thepharmaceutical composition comprises a pharmaceutically acceptableexcipient.
 11. The method of claim 1, wherein the pharmaceuticalcomposition is administered orally.